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Modelling the Substrate Binding Domain of Horse Liver Alcohol Dehydrogenase, HLADH, by Computer Aided Substrate Overlay
Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 17 © 1995 Elsevier Science B.V. All rights reserved.
479
Modelling the Substrate Binding Domain of Horse Liver Alcohol Dehydrogenase, HLADH, by Computer Aided Substrate Overlay Maija Aksela and A.C. Oelilschlager
6.1.
INTRODUCTION
Enzymes are catalytically active proteins that are involved in every in vivo transformation. They enhance the rates of biochemical reactions by 10^ to 10^2 by reduction of the free energy of activation J Two distinctive properties of enzymes are their high substrate specificity and the narrow range of conditions under which they are effective. They usually catalyze one reaction of a few substrates. Activities are dependent on pH, temperature, the presence of cofactors, as well as concentrations of substrates and products. Enzymes perform specific reactions because they possess cavities in which substrates are oriented while they are transformed (Figure 1). This process Involves interaction of the substrate with amino acids of the enzyme. Enzyme-substrate complexes have been studied by kinetic analysis, chemical modification, inhibition of enzymes by specific compounds that Interact with active sites, detection of characteristic spectral absorption bands during reaction of enzymes with substrates, and X-ray crystallographic analysis of enzymes combined with compounds which are in similar structure to the natural substrates. The Interaction between enzymes and substrates has been analyzed by the concepts of "lock-andkey" and "Induced fit". The former presumes that the substrate surface must fit the enzyme surface like a key In a lock, while the latter refined theory assumes that binding of the substrate induces conformational changes in the enzyme to provide a better fit. 6.2.
Enzymes as Catalysts in Organic Synthesis
The potential of enzymes as catalysts in asymmetric synthesis has been recognised for many years.2-12 Rate acceleration and stereoselectivity, together with techniques for the low-cost production and the rational alteration of their properties, make enzymes attractive as chiral catalysts in organic synthesis. Enzyme-catalyzed reactions have been categorised into six main groups''^ as shown In Table 1. Three of them, oxldoreductases, hydrolases, and lyases have been found useful In organic synthesis.
480 About 50 of the 200 enzymes produced industrially have been shown to be important to synthesis.I"* Active site
© Enzyme
Substrate
Enzyme-Substrate Complex
Product
E-Product
Figure 1. Schematic diagram of an enzyme-catalyzed reaction. Table 1. Enzyme classification. Class
Action
Oxido-reductase
Oxidation-reduction reactions.
Transferase
Transfer of a functional group (e.g. amino, methyl) from one substrate to another.
Hydrolase
Bond cleavage by addition of water.
Lyase
Reactions involving additions to double bonds or cleavages with formation of double bonds.
Isomerase
Ligase
Racemization of optical or geometric isomers and certain intramolecular oxidation- reductions. Formation
of
new
triphosphate (ATP).
bonds, cleavage
of
adenosine
481 The chemo-, regio- and enantloselectivlty of enzymes make them Ideal asymmetric catalysts. Most Important Is the differential recognition of diastereotopic groups of chlral and prochlral substrates. The mild conditions under which most enzymes operate minimize Isomerizatlon, epimerlzation and racemization associated with many other chemical processes.^.Q Maturation of the petro-chemlcal Industry, environmental pressures for "clean chemistry" and the explosive development of biotechnology have increased interest in the application of enzymatic processes to organic synthesls.3 Enzymatic processes play an increasing role In the generation of chlral pharmaceutical Intermediates, watersoluble materials and Copolymers. One problem In the development of enzymatic reactions for organic synthesis Is the prediction of the stereochemistry of reaction. Reliable models for prediction of stereochemistry are needed to broaden the application of enzymes to organic synthesis. 6.3.
Horse Liver Alcohol Dehydrogenase as a Catalyst
Horse liver alcohol dehydrogenase, HLADH, (also abbreviated as ADH or LADH) is the most extensively studied oxido-reductase. It plays a central role in ethanol metabolism and has been one of the main tools for understanding the mechanism of this process.'iS it was crystallized from horse liver In 1948 by Bonnlchen and Wassen and Is commercially available. Three Isozymes EE, ES and SS are formed by dimeric combination of two different, E or S (E "ethanol-active" and S "steroid active"), protein chains.''6 The EE- Isozyme of HLADH has been used in organic synthesis. HLADH Is nicotinamide coenzyme {NAD+/NADH) dependent and catalyzes the redox equilibrium between a large number of alcohols and ketones or aldehydes (Figure 2). The equilibrium is overwhelmingly in favour of the reduction reaction.^ Phosphate analogs of the coenzymes, NADP+ and NADP may also serve as coenzymes in very limited situations. The oxidation-reduction takes place through a ternary complex in which the substrate and coenzyme are simultaneously bound In the active site of enzyme. A zinc Ion at the active site binds the substrate and facilitates hydrogen transfer by acting as a Lewis acld.17 it has been assumed that the alcohol forms a zinc bound alkoxide during the reaction.is His-51 has been suggested to be important In transfer of a proton between the active site and surrounding solvent.''® Which amino acids are
482
9
HH..
R
H.N'Y^ OII '^ i A- --fNADH
NAD-"
NH2 HO
OH
^ O
O
^
OH
OH
N ^T^ll
^ 3 HO
f OH
Figure 2. Schematic view of the oxido-reduction catalyzed by HLADH. responsible for the observed pH dependencies of coenzyme association and dissociation as well as substrate binding and hydrogen transfer steps are not yet certain.19 The oxidation has been suggested to occur by an ordered mechanism in which the productive substrate binding site is formed after coenzyme binding. Dissociation of the enzyme-NADH is the rate limiting step.20 OH
f
CH3OH R
HO'
RCH2OH
R'
bnoH R'
d 6T Q5^
YADH HLADH Steroid alcohol dehydrogenase
Figure 3. Structural
specificity
of
HLADH
compared
with
dehydrogenase, YADH and steroid alcohol dehydrogenase.
yeast
alcohol
483 Over one hundred compounds have been examined as substrates for HLADH. It has a broad structural specificity and is well suited for organic synthesis. The enzymes reacts with acyclic, mono-, bi-, tri- and tetracyclic (steroidal) substrates. Figure 3 shows a comparison of the substrate specificity of common dehydrogenases.^^ HLADH has a wider use than yeast alcohol or steroid alcohol dehydrogenases. It operates on most substrates with high enantiomeric specificity. Examples in which HLADH exhibits superior selectivity include preparation of stereoisomers of bridged bicyclic
(±)
O unreactive enantiomer
83% e.e.
2.
100% e.e. H
H
(±) P
^OH
3. XH2CH2OH
'CH2CH2OH
(±)
4.
97% e.e.
J 10'''^ HO-^
L ^OH ^OH (±)
O
^
J^ J a'^^^ O'^'^X)-^ 100% e.e. OH
^ ^ unreactive enantiomer
"^ " ^
macrolide and polyether antibiotics
OH
5. (±)
100% e.e.
Figure 4. Examples of HLADH-catalyzed reactions.
100% e.e.
484 compounds (entry 1 in Figure 4).22 Stereospecific reduction of highly symmetrical diketones is also facile (entry 2, Figure 4).23 it is also possible to combine several different kinds of specificity to achieve in a single step a transformation that usually requires several reactions (entry 3, Figure 4).24 Chiral lactones, which are useful starting materials for many natural products, can be formed by stereospecific oxidations of mesodlols (entry 4, Figure 4).25 Even thioketones are reduced stereospecifically (entry 5, Figure 4).26 Reactions with HLADH typically occur at temperatures between 4°C and 25°C and in the pH range of 5 to 10. For catalysis of a reduction the optimum pH is ~7 while for the reverse oxidation it is -8.^ Reaction times vary from a few hours in the most favourable substrates and 2-3 weeks for the slowest. The disadvantage of HLADH has been the high cost of coenzymes.
Fortunately, several recycling methods are available that
allow reduction of substrates at the research scale (up to 1 kg of substrate).27-30 Por example, the ethanol-coupled method has been used for reduction and flavin mononucleotide (FMN) recycling for oxidation. 6.4.
X-Ray Crystallographic Studies of HLADH
Understanding of protein structure and function has been greatly enhanced by X-ray crystallography. At low resolution (4-6 Angstrom), the electron density map reveals the folding of the polypeptide chain, but few other structural details. At 3.0 A , it is possible, in favourable cases, to resolve amino acid chains, while at 2.5 A the positions of atoms may often be given with an accuracy of ± 0.4 A. In order to locate atoms to 0.2 A, a resolution of about 1.9 A and very well ordered crystals are necessary.3i.32 Three different crystal forms of HLADH have been studied crystallographically: orthorhombic (C222) crystals of the apoenzyme (native enzyme), triclinic (PI) or monoclinic (P2-|) crystals of the holoenzyme (apoenzyme with its coenzyme) and of ternary complexes (holoenzyme with substrate or inhibitor).''S j h e X-ray structure of the apoenzyme (EE-isozyme) was determined to 2.4 A resolution in 1976.33 The structures of the ternary complexes with NADH and dimethyl sulfoxide,^^ NAD+ and p y r a z o l e , 3 5 NAD+ and p-bromobenzyl alcohol,36 tetrahydro-NAD and dimethylamlnocinnamaldehyde37 and NAD+ and 2,4-(4-pyrazolyl)butylisoythiourea,38 have been determined to 2.9 A resolution. HLADH (EE) has been found to be a dimer comprised of two identical subunits of Mr 40,000 (Figure 5). Each subunit contains both a coenzyme and a substrate binding
485 domain. The single polypeptide chain of one subunit contains 374 amino acids of known sequence.^d These two subunits are separated by a cleft, containing a deep pocket. Both subunits of the dimer bind coenzyme and substrate in essentially the same manner; thus these two active sites are assumed to be the same.^s The two subunits of the dimer are linked by interactions within the coenzyme binding domains which form a core of 143 residues in the middle of the molecule. The catalytic domains are at opposite ends of the dimer and comprise 231 amino acid residues.33
Figure 5. Schematic of dimeric HLADH.^^ The coenzyme binding domain has a structure very similar to corresponding domains in other dehydrogenases and some kinases. The coenzyme binds in the cleft across the edge of the coenzyme binding domain via several hydrogen bonds, charged and hydrophobic interactions.'^^ The nicotinamide moiety of coenzyme is positioned in the active site with the A-side of the ring facing the zinc atom.^^ Coenzyme binding induces a conformational change from an open to a closed form. The enzyme may exist in both conformations, but the equilibrium favours the open conformation in the absence of coenzyme binding.i^
486 The major conformational difference between the open and closed forms is a rigid body rotation of each catalytic domain by about 10° with respect to the coenzyme binding domain. Thus, a cleft between the catalytic and coenzyme domains is closed, making the active site less accessible to solution and more hydrophobic. During this change some amino acid side chains, especially that of, Val-294 change their orientation. The side chains of Leu-57 and Val-294 which are about 15 A away from each other in the apoenzyme form, are only 4-5 A apart in the ternary complex. No new residues are brought into the substrate binding domain. The conformational change also brings the catalytic zinc 1 A closer to the nicotinamide binding domain and moves the substrate and nicotinamide binding domains closer. The interactions between the protein and the nicotinamide ring of coenzyme are assumed to be involved In Inducing the conformational change of the enzyme during coenzyme binding.18 COENZYME
174
CYS
Figure 6.
48
Schematic of ethanol in substrate binding domain of HLADH.
The substrate binding domain has two zinc atoms, one of which, the catalytic zinc, is located at the bottom of the hydrophobic substrate binding domain, 20 A from the surface of the molecule. Cys-46, Cys-174 and His-67 are ligated to this zinc which binds the substrate in a position relative to the coenzyme that facilitates direct hydride
487 transfer.""^ The function of the second zinc atom and the protein lobe that surrounds it, Is unknown. A deep pocket accommodates the substrate and the nicotinamide moiety of the coenzyme. It is 5-10 A wide and --20 A long, extending from the protein surface to the catalytic zinc atom. The part of this pocket associated with substrate binding in HLADH Is much larger and more hydrophobic than in other dehydrogenases. The substrate binding domain consists of a large hydrophobic barrel lined with non-polar side chains of Leu-57. Phe-93, Phe-110. Leu-116, Phe-140. Leu-141, Pro-295, Pro-296, lle-318 and Val-294, which are in the same subunlt as the ligands to the catalytic zinc that resides at the bottom of the barrel.33 The only polar groups in catalytic site are Ser-48 and Thr-178 (Figure 6). In the open apoenzyme form several firmly bound water molecules are located in the inner part of the pocket outside the co-ordination sphere of zinc. In the closed form of the ternary complex the coenzyme and the substrate occupy this space.''S According to the X-ray crystallographlcally determined structures of ternary complexes of HLADH the side chains of amino acid residues bordering the substrate binding domain can adopt chain conformations compatible with the volume occupied by the bound llgand. In the binding of p-bromobenzyl alcohol and the inhibitor, dimethyl sulfoxide, there are changes in the positions of the side chains of Leu-116 and Ser48.36 The conformation of Leu-116 is also different in the NAD+-4-lodopyrazole and (dlmethylamlno)cinnamaldehyde complexes.37 in the NAD+ and 2,4-(4pyrazolyl)butylisothlourea complex side chains of Met-306, Leu-57, Leu-116 and lle318 assume different conformations than in the HLADH-NADH-dlmethyl sulfoxide complex.38 6.5.
Previous Models for the Substrate Binding Domain of HLADH
Several models have been developed for the substrate binding domain of HLADH from kinetic studies and/or X-ray crystallographic data. The substrate binding domain has been analyzed using diamond lattice and cubic section models (Figure 7). The first attempt to describe the topology of the substrate binding domain was by Prelog In 1964 In terms of the diamond lattice model.^"' This work was refined by Jones et al. In 1976 and 19778.42,43 and Horjales and Branden in 1985.^4 The first cubic section model of HLADH was developed by Jones and Jacovac in 1982.'^5 other types of
488 models have been developed by Ringold et al. in iges,'*^ Nakazaki et al. in 1980,^-^ Duller and Branden in 1981^8 and Lemiere et al. in 1982 (Table 2)."^^ The diamond lattice model (Figure 7) was developed using six-membered ring ketone substrates. The determination of forbidden and undesirable positions was achieved by analysis of the relative rates of reduction of a series of cyclohexanones and decalones of known absolute configuration.8 The geometry indicated at the C-0 centre was considered to resemble the structure of the alcohol rather than that of the ketone in the transition state. It was assumed that all substrate molecules bound with oxygen in the
("-
\\A] O — undesirable position # - forbidden position
0
OH - forbidden cube
Figure 7. Diamond lattice and Jones cubic section model. same orientation and direction of the forming C-H bond. The back of the lattice section was thought to be a flat coenzyme binding site and the lower and left sides of the lattice were thought to be bounded by the enzyme. The oxidation and reduction was assumed to be forbidden if binding of a substrate superimposed a group in one of the enzyme occupied locations. If the group occupied an undesirable location, the rate of reaction would be very slow. Horjales and Branden (1985) constructed a diamond lattice model by docking cyclohexanol and its monoethyl derivatives into the experimentally determined active site of the enzyme (X-ray crystallographic structure, 1982), using computer graphics and energy minimization methods."*"^ The lattice positions were classified as allowed, forbidden or boundary depending their distances to protein atoms (Figure 8). The
489 lattice positions were considered as allowed (A), if all distances to neighbouring atoms are larger than 2.9 A, forbidden (F), If any distance to a fixed atom Is less than 2.6 A or boundary (B). Boundary regions were sub-grouped into B 1 , consisting of those positions that Interact only with side chains of Leu-57, Leu-116 or Met-306 and B2 comprising all other boundary positions. The volume available to substrates was similar to the substrate binding domain derived from analyses of previous kinetic data of the methyl cyclohexanones (Dutler and Branden, 1981^^) and the diamond lattice model (Figure 9a).s Using this method, Horjales and Branden extended the lattice model to extend the limits of definition of the substrate binding domain (Figure 9b). Table 2.
A summary of previous models and methods HLADH, presented
chronologically Author
Year
Models
1. Prelog
1964
2. Ringolde/a/.
1965
3. Jones et al.
1976
4. NakazakI ef a/.
1980
5. Dutler & Brandon
1981
6. Jones & Jacovac
1982
7. Lemlereet al.
1982
the diamond lattice model, using kinetic data composite structures of satisfactory and unsatisfactory substrates In front and side views the refined diamond lattice model the C2 -ketone rule oxidation and reduction cyclohexanol rings superimposed and compared; kinetic data; X-ray of apoenzyme the cubic section model (manual) "flat" cyclohexanone model
Substrates cyclohexanone and decaione derivatives 3- and 4-alkyl cyclohexanones, 10methyl-2-decalones cyclohexanone and decaione derivatives the cage-shaped C2ketones 2-,3-,4-alkylcyclohexanol with alkyl groups, methyl, ethyl, l-propyl and t-butyl. alkyl cyclohexanones 2-,3-,4-alkyl cyclohexanones
8. Horjales & Branden 1985
the diamond lattice
cyclohexanol and Its
model, using docking
monomethyl derivatives
& computer graphics
490
asfi(\
utnoi
Figure 8. Stereo diagram of the substrate binding domain using the substrate docking method.
Figure 9. Figure 9a: A comparison between two diamond lattice models. Positions A-J are derived by kinetic studies as forbidden or hindered. Positions K-R represent boundary or forbidden positions defined by Horjales and Brandon. Figure 9b shows the extended diamond lattice model. In 1982 Jones and Jacovac mechanically constructed a cubic section model using Framework Molecular Models.-* The cubic section model was conceived because
491 substrates other than those containing a cyclohexyl ring, e.g. cyclobutanones, and cyciopentanones were not easily fitted to the diamond lattice model. The lattice was based on sp^^-hybridised carbon lengths and angles. Thus, accurate predictions for numerous substrates were difficult to make. Models that resemble the cubic section approach have been developed for mono-oxygenases (Johnson in 197850) and microbial reductases (Nagazaki etal. in 198051). Jones and Jacovac employed the kinetics of alkyl cyclohexanone reductions (Duller et al. 1977,1981 and Lemi^re et al. 1980) for the estimation of forbidden and limited access volumes. In addition, they used the X-ray derived structure of the apoenzyme, the enzyme-NAD complex and several ternary complexes to derive the shape of the substrate binding domain (Figure 10a). The model, used 1.3 A3 cubes and the C-0 function is defined as an alcohol as in the diamond lattice model. The oxygen of substrate Is assumed to ligate to the catalytic zinc and hydride delivery or abstraction occurs from the front. Jones and Jacovac defined forbidden and limited access regions as A1-2, El-3, G4-5, L4-6, M7-8 and Q7-9, the left-hand halves of B1, H4 and J4, P7 and the right-hand halves of B1, H4 and N7 as well as cubes 0 9 , P8, P9. Binding in the limited access area results in a reduced rate of reduction of substrate. Cubes below the plane of the paper reflect the character of cubes immediately above them. The allowed region was suggested to correspond to the substrate binding domain of HLADH as identified by X-ray (Figure 10b). However, the boundaries between the allowed, limited and forbidden regions were assumed to be flexible due to the movement of amino acid side chains during substrate binding. The forbidden cube
y/ipg/^ y^ y%
**.^T"" "^y^
[M
N
o
p
Q
R
G
H
1
J
K
L
A
B
0
D
E
F,-- ..t.
5
>
-aC
^^
U
Figure 10.
U(F1)
£9
8
/ 4
2
»
3J
V
Leu57 Ser 48
\Z^ ^
OH
Figure 10a, Jones cubic section model. Figure 10b, Correlation with X-ray crystallographic studies.
492 E1 corresponds to the Phe-93 location; K4 and Q7 to lle-116; E2, E3 and K5 to Phe110; 0 9 , P9 and Q9, to lle-318; B1 to Ser-48; G4, M7 and half of the cubes, H4 and N7 to Leu-57 while J4 and P7 are limited because of the nicotinamide coenzyme. O
6 (2R)-]
,"^2- tt::p=^ — H - ^ : i i j 2 : i £ ! i ^ o I N7
07
P7
Q7
H4
14
J4
K4
B^
El
B1 >^1
KJ-^H
O
II
HD' ^ H
6' (2S)-1
HLADH (2S,2S)-2
I
iV^^X
Figure 11. Prediction of stereochemistry of reduction for 2-alkyl cyclohexanones by Jones cubic model. The direction of hydride delivery from NADH is indicated by the arrows > and > for reactive and unreactive product or orientations, respectively. Only conformation IV can bind without penetrating forbidden regions.
493 The prediction of product fit into the HLADH active site was conducted separately for each enantiomeric product, using the mechanical model. For the product to fit well, the bound substrate must not occupy forbidden positions. Penetration of substrate Into two or more cubes of limited access was judged to be equivalent to a forbidden interaction. The Jones group have applied this model to more than 50 different cyclic and bicyclic compounds (e.g. Figure 11). The predictions for both reductions and oxidations use the same model and are reliable. 6.6. Present Cubic Section IVIodei for HLADH 6.6.1.
Generai
The goal of the present study was to develop a computer-based cubic section model of the substrate binding domain of HLADH. It was considered that the Jones cubic section model could be refined by use of computer assisted substrate overlay in combination with kinetic data on a wide variety of substrates. As in the Jones approach we used the alcohol products as the surrogate substrate structures. Thus, we determined the low energy conformation of alcohols produced from ketones that have been reported to be reduced by HLADH and for which comparative kinetic data vs cyclohexanol could be calculated. As well, we determined the preferred conformations of all alcohols that would have been produced from ketones subjected to but falling to undergo HLADH reduction. These calculations utilised molecular mechanics (MACROMODEL) and yielded accurate co-ordinates for all atoms in each alcohol. Where enantiomeric or stereoisomeric alcohols were produced or capable of production, the co-ordinates of each were calculated. Alcohol products were assigned a priority number based on their rate of production vs cyclohexanol. Relative reduction rates, enantiomeric excesses of alcohol products, extent of conversion of substrates, yields and absolute configurations of alcohols were used In calculating the priority number for each substrate with the faster reacting substrates receiving the higher priority numbers. Co-ordinates of atoms in the energy minimised alcohols were transferred to ENZYME, a program that allowed the structures to be identically oriented in a cubic section model. Using ENZYME program the energy minimised substrates were placed in a Cartesian co-ordinate (x, y, z) system with C-0 aligned with the carbon at the origin, the oxygen on the negative y axis and the ahydrogen the yz-plane.
Conducting this operation on all alcohols gives a map of
acceptable and forbidden cubes according to the average priority numbers for each
494 cube. Forbidden areas were assumed to be occupied or blocked by amino acid residues or coenzyme. The model is directly comparable to that of the Jones group in that we have used cubes of 1.3 A on a side and placed the carbon-oxygen bond as well as the carbon-hydrogen bond of the carblnyl carbon In the same orientation. We have compared the present model with that of Jones as well as with the X- ray crystallographic structure of the enzyme. 6.6.2.
MACROMODEL and ENZYME Programs
MACROMODEL and ENZYME allow facile structure entry, energy minimization, reorientation, atom to cube assignment and calculation of average cube priority values for each cube.
MACROMODEL, developed by Clark Still at Columbia University
employs a recent version of Allinger's popular force field minimization routine. Each molecule is described in the program according to the atom types It contains and the connections between atoms. The energy of each conformation of each structure is calculated by evaluation of the Van der Waals, bond stretching, bond angle bending, torsional and dipole interactions. In Appendix 1 there is a description of those parts of MACROMODEL that were useful in this work. ENZYME SITE PREDICTION SYSTEM V 1.6
• • Bi m •
-
Forbidden Priority < 0.5 Priority < 1 Priority < 5 Priority < 10
•
- Priority >= 10 Layer 1
Cube file is col 3.cf
READ CF READ DATA ADD TO CF REMOVE CF ALIGNMENT ROTATE ON X ROTATE ON Y ROTATE ON Z PREDICT DISPLAY CF ROTATE BONDS
Press
PRINT CF SAVE EXIT CSl.30 CORNER DISCOL 6
Figure 12.
Menu of the ENZYME program. One layer of constructed substrate
binding domain is presented.
495 The ENZYME program was developed at Simon Eraser University In Pascal on a VAX 750 using an IBM personal computer with an enhanced graphics adapter and graphics tablet running a VT 100/Tektronix 4107 terminal emulator. The source code program is available from the authors. It is fully compatible with the MACROMODEL program and was designed to be easily transferred between computers. ENZYME allows construction of cubic section substrate binding domains for enzymes other than HLADH. The program executes various procedures through menu driven processes. Representative procedures are shown in Figure 12 are read the product file, add the information for the molecule to the cube file, remove information for the molecule from the cube file, align the molecule in space, rotate about an axis, predict the averaged priority for the product, display cube file data, rotate about a bond, change the size of the cube, shift the origin from the center of the cube to the corner, print the cube file data, save the cube file and exit the program. An additional program, COMPARE, allows plots and cube by cube comparison of two constructions of substrate binding domains. COMPARE was also written in Pascal on a VAX 750 using an IBM personal computer configured as above and the source code is available from the authors. The documentation of ENZYME and COMPARE menus are presented in Appendix 2. The details of the use of the programs, MACROMODEL and ENZYME are described in Chapter 7.5. 6.6.3. Criteria for Choice of Substrate Surrogates As in the Jones protocol the cubic section model of the substrate binding domain of HLADH were constructed using structures of alcohol products rather than ketone substrates. The alcohol products were originally chosen by the Jones group because the transition state the geometry for the reduction was considered to resemble that of the alcohol rather than that of the ketone. The relative rate of reduction of substrate vs cyclohexanone for each ketone was required to be known. Furthermore, configurations of alcohol products, enantiomeric excess values, yields and % conversion of substrate required for calculation of the priority number for each enantiomer of product should be measured under comparable conditions (i.e. pH, temperature, concentration of enzyme, coenzyme and substrate, etc.). According to Alderweireldt et al. (1988) HLADH models are valid only for reaction conditions used in the reactions from which the models are constructed.52 Furthermore, the model is only reliable if the reactions have been conducted under kinetic control.
496 Table 3. The substrates used in construction of the active site picture. OH
OH
OH ,
OH ,
^48.53
2^.53
38
OH
OH
OH
g26
754
g54
OH
OH
^353,55
^^53,55
OH
OH
^48
5S4
g54
OH
OH
OH
,0^
„26
^^26
OH
cis & trans
cis & trans
^^
trans
2 1 ^ ' 22^=*
23^'
cis & trans
,ge
-.8 ,78
cis & trans
,«8 ^g8
^g„
2o„
OH trans 24"
2 ^
2 ^
05 OCT A„ ^ 27®
32
28®
29^
33
34
X"
30^^
31®
35
36 OH
OH HO
^
HO 37
38
39
57
40
41 OH
^ 4 OH
OH
42"
43
,58.56
OH
44
45^
497 Using these criteria 45 substrates for which experimental information was available for reaction at pH 7 were chosen to construct the model (Table 3). There is a significant amount of literature data for substrates of HLADH which lack information on enantiomeric excess values and absolute configurations of products and where the relative rates of reaction have been measured under different reaction conditions (e.g. pH 8.5). Substrates falling Into this category (46-69, Table 4) were not used. Heterocyclic bicycllc substrates 70-73 were only used for testing.
O
Example 1: (±)
OH OH
O
Example 2:
OH
OH
ir &
^ (±)
OH
OH OH
X::^oH^^^
\::^
^=^/X:::poH
Figure 13. The possible conformations of two substrate surrogates.
498 Table 4. Product structures used in testing model for prediction of stereoselectivity. OH
OH
OH
OH
OH
46^^ 46''
4/^ 47'
48^
49^°
50^°
OH
OH
OH
OH
OH
6 ^ 6 ^ O^ Cx. 6^ .60
60
52'
51^ OH
53
OH
6
OH
.60.49
g^60.49
62
OH
^
-OH
^60.49
^H
6< 6: A^.o^ 67^
66"
HO"^-^^
71
68"
HO-^^T"^
72'
69"
70"
HO-^^-^TT"^
73'
Structurally rigid substrate surrogates were added to the cubic section model before more flexible molecules as the following order signifies: pentacyclic, tetracyclic, tricyclic, bicyclic ketones, trans/cis-decalones, methyl cyclohexanones and alkyl cyclohexanones. The hydroxyls of cyclohexanols were oriented axlally with respect to cyclohexyl rings consistent with the Jones protocol and with the observation that
499 reduction of conformationaily locked trans-decalins (16,18, 20, 22 and 23) produced exclusively axial alcohols. All low energy conformations of conformationaily flexible alcohol products were added included (Figure 13). 6.6.4.
Calculation of Priority for Alcohol Products
The priority number for an alcohol product was determined by the relative rate and the stereoselectivity of reduction. The priority number for each substrate surrogate was calculated from the rates of production of each enantiomeric alcohol product using the total relative rate of reduction vs cyclohexanol and the enantiomeric ratio (E) formula derived by Sih (Table 5).^^ Table 5. Formulas for calculations of the priority numbers for product enantiomers.
ln[1-c(1-fee(P))] *=~ln[1-c(1-ee(P))]
Sih's formula For product 1:
(1) (2) (3)
For product 2 :
P+ = R ( Y , + Y 2 ) ^ E 2 + I )
p-=f^(v^)(E?n-) where
(4)
(5)
E = the enantlometric ratio, the discrimination between two competing enantiomers by enzyme c = the extent of conversion of racemic substrate e e ( P ) = the optical purity, the enantiomeric excess of the product P = priority number for major enantiomer p = priority number for minor enantiomer Y = yields of products R = relative rate vs. cyclohexanone
The relative rate for cyclohexanone reduction was set at 100. This would give, for example, a total relative rate of 24 for the reduction of 2,3,5,6-tetrahydro-2-
500 isopropylpyran-4-one (precursor of 7, Figure 14)7 ENZYME contains a subroutine to calculate the priority numbers. It will request ail necessary values for the equations (Table 5). The ratio of enantiomers (major or minor) are to be given by the user. The priority values are calculated and displayed by using the PREDICT opWon of the menu. The priority number, 22.6 is given for the major product, (2S,4S-7). For the enantlomer, the priority number is near zero because the enantiomeric excess for this reaction is 1.00 (Figure 14). OH
QH
0.r
R = 24
c = 0.5 (2S,4S) Yi=32 ee=1.00
(2R,4S) Yi=2 ee=0.28
(2S.4S) Product 1:
P+ = 22.6 E = 15200
'
(2R.4R)
p. = C
(2R.4S) Product 2:
E = 2.3
I
P+=1.0
(4)
p. = 0.4
(5)
>
(2S.4R)
Figure 14.
(2)
^
Priority number calculation for enantiomers of 2,3,5,6-tetrahydro-2-
isopropyl-pyran-4-ol.^ 6.6.5.
Construction of Substrate Binding Domain for HLADIH
A global energy minimised conformation using MACROMODEL was obtained for each compound to be used In construction of the model. The torsional angle constraint was set at zero degrees for the atoms needed in this orientation then the minimization process was executed using the Block Diagonal Newton Rapson minimization method, BDNR.
Compounds to be included in the model were reoriented according to the
Jones protocol, using the MACROMODEL ANALYZE mode (ALIGN X, ROTATE Y options). The C-0 bond is aligned with the carbon at the origin, the oxygen on the negative y axis and the a-hydrogen in the yz -plane (Figure 15). The coenzyme is assumed to be in front of the origin and the catalytic active zinc atom is in the lower portion, directly ligated to the hydroxyl group of the substrate.
501 Each compound data file was transferred in ASCII format from MACROMODEL to the ENZYME program by execution of MM FORMAT on the file produced by MACROMODEL. Compound files in ENZYME are called for entry Into cube files by using READ DATA option. At this point the program requests the priority number of the compound which was calculated by the process, explained in Section 7.4. Using the ALIGNMENT option each compound was aligned with reference to the axes with the carbon on the origin (0, 0, 0). It was helpful at this juncture to check the orientation of each structure by rotation about the y-axis by ±10 degrees. Correctly minimised and oriented structures were added to the cube file of a chosen name using the ADD CUBEFILE option. After each stmcture was added the cube file was saved. The model produced by this process could be viewed, using the DISPLAY CUBEFILE option. To obtain the co-ordinates of the cubes and average priority value of each cube the PRINT CUBEFILE option was used. Addition of a structure to an empty cube file assigns each occupied each cube of the model the calculated priority of the structure. If atoms of a second structure occupy some previously marked cubes the average of the priorities for the entered structures will be calculated for these cubes. When a structure known not to be produced (i.e. Its priority is zero) is added to the model all cubes not previously identified as being occupied and which are occupied by that molecule will be marked forbidden. The forbidden classification will be removed If a subsequently added structure has atoms occupying those cubes. The program does not use priority values of zero to calculate average priorities. Addition of many structures to a cube file creates a cubic section model in terms of allowed (with averaged priority numbers) and forbidden (priority 0) cubes. Each cube is identified according to the Cartesian co-ordinates (x, y, z) of its corner farthest from the origin. The modelled volume contains several layers of cubes of user defined size. ENZYME allows average priority values of cubes to be viewed in different colors (represented as shades in Figure 12). Display of cube priorities in two colors can be chosen. Green is the default color for allowed cubes and red for forbidden cubes. A hard copy of the average priority values for each cube can be obtained by using the PRINT CUBEFILE -menu option. The method allows openended improvements since new structures can be added to refine cavity topology.
502
^
Reaction
_^h^£L,
J^
(±) rel rate vs cyclohexanone = 26 conversion of the reaction * 0.46 an enantiomeric excess of product = 0.64 [^ a yield of product = 39
E - 7.76
Computer simulation H
(0-^
MM2
A T H -^--'- y^H
DRAWN
I
PRIORITY = Acceptance ranking
REORIENT
OH 2.97
23.03 (01lY
I
yo
z:
m
I cube - atom match P = cube acceptance ranking
cube acceptance ranking = P
A = average acceptance ranking
I repeat for additional substrate Figure 15. Procedure of defining topology of the substrate binding domain of HLADH.
503 Using the same equivalently oriented structures 12 different cube files were constructed (Table 6). The effects of location of the origin in the corner or In center of the center cube, size of cubes (1.3 A or 0.65 A) and inclusion of hydrogens in the structures were studied. Special cube files, coded co13*, co065*, ce13* and ce065* were constructed without the hydrogen on oxygen (Table 6). Usually this hydrogen can occupy two side by side cubes in layer -1. This is due to very slight differences in orientation produced during the minimization process. Table 6. Twelve different cube files constructed. orii jjln corner center
Icode of cube file
1. co13
X
2. CO065
X
3. 0013*
X
4. CO065
X
5. co13h
X
6. co065h
X
X
X
X
X X
X
X
X
11. ce13h
X
12. ce065Hi
X
X X
X
X
10. ce065*
6.6.6.
X X
8. ce065
1
X
X
X
X
a-H&H on SL b s t r a t e s oxygen g-H aii hydrogens
X X
7. ce13
9. e e l 3*
cub ) size 1.3A 1 0.65A
X X
X
X X
X X
X
Model for Substrate Binding Domain of HLADH
A goal of this work was to refine the cubic section model developed by Jones"^^ using computer modelling. Thus, equivalent orientations of compounds, cube size, 1.3A. and origin location were used to construct the cubic section models of HLADH. Because the jacks and plastic tubing of the mechanical model occupy space, Jones changed
504 the edges of cubes C, D, I, and J in layer 0 to 1.4 A. Computer modelling allowed the Present Model (co13) Jones Model
layer 4
layer 3
layer 2
M^ N
0
P
Q
R 1
p
H
1
J
K
L
r
B
c p
E
F
u
u
u
u
u
u
1
505
layer 1
[N
0
H
1
B
c p
u
U
^M J
u
layer 0
0.02
^ ^
1
layer-1
4
34
1
lofH
I1
45 10 9 11
506
layer-2
fA
N
0
P
Q
R1
r
H
1
J
K
L
r
B
c p
E
F
u
u
u
u Cubes:
u
u
n
•
|58j allowed in present model I
>
(5 = averaged priority number, 8 = number of atoms)
I allowed in Jones model forbidden
1 1
cube size 1.4 A
forbidden or limited in Jones model limited in Jones model allowed for some 3-alkylcyclohexanols or heterocyclic bicyclic alcohols (usually forbidden) allowed for some 4-alkylcyclohexanols (before unknown) Figure 16. Cubic section model for the substrate binding domain of HLADH: Jones model (left) and the present (co13, right). Jones assigned each cube alphabetically and we have used his convention to allow comparison. The priority numbers for each cube were rounded in the present model. size of ail cubes to be of the same. The construction of the original cubic section model used only the kinetic data of alkyl cyclohexanones. In the present model 166 different conformations of alcohol products and potential products were used to construct each cube file. Conformations of 55 had positive priority values, while 111 represented non reactive conformations.
In both the Jones and the present models substrates
contained only one carbon bound hydrogen which is the a-hydrogen and a hydrogen on oxygen, in our model other hydrogens of modelled structures were deleted after minimization. The previous division of cubic space into allowed, limited and forbidden layers spaces has been refined in that ENZYME generates individual acceptability rankings for each cube (Figure 16). ENZYME gives a printed list of the cube file: the contents of each cube, the average priority of cubes and the total number of atoms eventually in each cube. For model c o l 3 the origin is in cube OD (cube D in layer 0),
507 the oxygen In cube OD-2 (2 cubes under cube D in layer 0 ), the a-hydrogen In cube -1 D (cube D In layer -1 ) and the hydrogen on oxygen In cubes -1C-2 or -1D-2. In both the Jones and col 3 models the allowed area opens most significantly to the left of the origin. This can be seen more clearly by the present model because layers 3
Table 7. Comparison between cubes in Jones and col 3 models. 1 layer, 1 the code Jones model Present model the contents of cube of cube col 3 & priority ( ) layer -1 H K 0
F F F
34 4 0.02
substrate 30a(39) 30b(4) 31(0.02)
layer 0 G M N H e J Q K
o aM-aR 1
F F F&L F&L L L F F A ?
0.2 0.8 0.01 1 2 12 1 1 0.2 17 1 For A*
! 1
15a(0.2) 2b(0.57).14a(0.98) 45b(0.01) 10 substrates 33d, 41b, 90a. 30b 15 substrates 45a(1.29) 38d(0.27),37d(0.05) 38c, 30a *aO: 70a
layer 1 A & uA G M H N uB o
J p Q K E uE F—2 aN-aQ
F F F A A AorL A A. F or L F F F F F ?
For A* 0-2 unk 2 2 2 0.5 2 5 4~ 0.1 13 1 0.4 For A*
*52a, 53b, 55a, 48a I5a(0.2) 38a, 37a, 40a 37c. 41b, 44b 12a(3.6),39b(1.01) 37c, 37d, 42b 11 substrates 37c, 37d, 42a 44a(94.9),37d(0.27) 38b(0.08),37b(0.1) sulphur-compounds 39a(1.49) 7d(0.43) VO to 73
1
508
layer 2 K
F
Q E
F F
uE
F
uA bG uC B-2 N G 0 P
A A AorL A A A ForL ForL
0.4 unk 2 0.2 2 2 1 F A* 13 4 For A*
45a(1.49),45b(0.57) 38d(0.87),37d(0.05) 38b(0.08). 44b(5.13) 45b(O.Oi) 7d(0.43), 6d(0.08), 5d(0.26) 2x120(2.39) 2x120(2.39) 39b(1.01) *58 110(23.2). 120(2.39) 17a(7.5). 41b. 19a 60a, 63b. 66b, 65a
layer 3 bG bA A B
? ? ? ?
61 61 31 11
C D C-2 D-2 D-l C-l J 1 aP aQ E
? ? ? ? ? ? ? ? ? ? ?
3 4 28 28 A* A*
1
1
A* A* A* A* For A*
8a(120.5), 120(2.39) 8a(120.5). 120(2.39) IOo(39.3). 110(23.2) 18a(3.0), 6a(14.8), 7a(22.6). 20a(3.5) 18a(3.0), 20a(3.5) I9a(4.0) 80(27.53) 80(27.53) *58a *59a 58a, 58b. 59a, 59b 58a, 59a 66b, 65a, 63b 65a 70-73
j
layer 4
1
4c
?
A*
61a. 63a. 65b. 66a
and 4 are more fully defined. Due to the greater number of compounds used the allowed area in the present model is slightly larger than in the earlier version. A list of average priority values for eaoh oube are given in Appendix 3. comparison of each cube in the two models is presented in Table 7.
The detailed
509 The cubes with priority numbers of 2 or lower are considered boundary areas (Table 2). They are allowed for some substrates and forbidden for others. The priority of structures that occupy these cubes is usually very low. Some bulky structures were found to occupy low priority cubes G, N, Q and K in layer 0, cubes G, K and uE in layer 1 and cubes K and E in layer 2. These are forbidden in the Jones model. Bulky substrates are assumed to change the orientation of amino acid side chains lining the substrate binding cavity. It is suggested that except for bulky structures these cubes will be forbidden as suggested by Jones. The high priority value of cube 1E (13.0) is caused mainly by sulphur containing substrates. In every case C2 of these heterocycles affects this cube making it allowed. The high value of cube 1Q (48.0) is due to one cage-shaped substrate (44a) with a priority value of 94.9. Another substrate (37d) occupying the cube has a priority value of only 0.27. It is suggested that this cube also belongs in a boundary area. Cube, OM, forbidden in the earlier model, is a boundary area with average priority value, 0.8 in the present (col3) model. The cube contains two atoms of 2-ethyl cyclohexanol (2b) and its sulphur analog (14a). Cubes 1H, 1N, 10 and U are allowed in the earlier model and each have an average priority value of 2 or lower 0013. They are occupied by atoms of bulky substrates. In the earlier model cube OJ was limited access, while In the present model its average priority value is high (12.0) and it contains 15 atoms of as many substrates. Cube IP, with a priority value of 5 (occupied mostly by bulky substrates), is a boundary area In both models. Cube OO, with a priority value of 17 contains bulky substrates (38c, 30a). Usually areas above cubes OM to OR are forbidden in col3. This volume was not defined In the Jones model. According to the Jones model regions below the origin reflect the character of cubes immediately above them. Thus, those cubes below limited or allowed cubes were assumed to be limited access. Cubes, 2D-2, 2D-3, 2E-2, 2E-3 and 2E-4 should then be limited although they are forbidden in col 3. The average priority value for cube 1M is undefined with the data we used. It could belong to a boundary area as does the neighbouring cube IN. Cube 2B-2 could be allowed if more data were available. Cubes 1F-2 and 2uE have low priority values and are suggested to be in a boundary area. Bridged substrates, 30a, 30b and 31 effect some cubes in layer - 1 , making them allowed. It is possible that these substrates change the orientation of amino acid side chains or coenzyme near this layer to allow binding. The cubes 3bA, 3bG, 3C"2, 3C-3,
510 2bA. 2A-1, 2C-2, 2C-3, 2B-3, 1B-2, 1A-2 and 1A-3 are occupied by atoms of phenyl groups of one or two substrates (8,12, Figure 16). Some structures were examined which were not included in the cubic section model used for testing. For example, linear and branched 3-alkyl cyclohexanols can change some forbidden cubes in col 3 to allowed. Also 4-alkyl cyclohexanols were found to extend the allowed area In layers 2, 3, and 4. These substrates were not used in the construction because they were studied under different reaction conditions or no values of enantiomeric excess were available.
Addition of substrates containing
hetereocyclic bicyclic rings (70-73) would change cubes OaO, 1aN, 1aP, 1aQ, and 3E to allowed (Figure 16). After construction of model col 3 wherein the origin was on the corner of a cube It was of interest to visualise the effect of placing the origin on the center of a cube (eel3, Figure 17).
This process was executed for the same set of structures used for
construction of the corner centred cubic sector model. Placing the origin at the center of a cube alters border areas and changes the average priority values on most cubes as well as the number of substrate atoms in each cube. Appendix 4 gives the printed list of cube priorities for the center cube origin model (ce13). The effect of cube size was also studied. Thus, the cubic section model was constructed with the same set of substrates as model co13 assigning the origin to the corner of the center cube but with a cube size of 0.65 A. The printed list of the cube priorities for this model (co065) is in Appendix 5. Altogether 14 different layers were defined. A combined view of both co13 (1.3 A) and co065 (0.65 A) models allows refinement of the 1.3A model (Figure 18). For example, only half of the cubes OK, OH, IK, I E , 2E and 2K allowed in the 1.3A model are only allowed in the 0.65 A model. The cubes OH, ON, I N , and 1H are regarded to be on a boundary area. Jones assumed that the right half at the cubes OB, OH, and ON were limited and half were forbidden. The present analysis allows clearer definition of the border of forbidden and boundary areas.
511
3.3^
o 28'
layer 5
p
61^
layer 4
23«
6^
3
61^
r
21
6l2
18«
l'
4^
5*
0.01
23^
0.22|
10^
"^
4'^
15 1 11^= 3='
iiH 9«
o
71 12H 4^
12 1
^^0.24^
2
4^
16
4'
16
2
28'
layer Z
layer 3
^ 4 11 ^0.5 11 0.5= 52 ^
1
11
12 5 8 4 0
15 6
p ^
3 P
11 J2
1 4
layer 1
layer 0
512
59
O
iQ> .55 10
layer-2 layer-! I
I allowed
^ ^
forbidden
o=origin
Figure 17. Cubic section model (ce13) of the substrate binding donnain of HLADH.
0
layer 4
layer 3
513
•ii
layer 2
layer 1
layer -1 layer 0
allowed forbidden
D
forbidden position in 0.65 A map
unknown
layer -2 Figure 18. Cubic section models of the substrate binding domain of HLADH using 1.3A (col3) and 0.65A (co065) cubes.
514 6.6.7.
Testing of the Models
All twelve cubic section models listed Table 6 were tested to determine which constituted the best model for prediction of substrate reactions with HLADH. The effects of the origin placement, cube size and structure of substrates were tested. In some analyses compounds that had been previously used in construction of the models were used. If this were the case the entries relating to these products were individually removed from the model before a prediction was executed. For new products the minimization, orientation and alignment processes were conducted before execution of a prediction. The PREDICT option (mouse driven) gives the average priority of cubes occupied by each atom of the test molecule and identifies those atoms in cubes of undefined and zero priority. PREDICT (pressing the second button of the mouse) will write the above information to a file with the same file name as the molecule file but with the extension '.pre'. Another way to visualise the results is to use the cubefile graphic display. The positions of each atom of the test molecule are seen as white circles in the individual layers of the model. The results of prediction process were analyzed using both the data in the prediction table (average priority value of each cube, average priority value of cubes over molecule, forbidden sites and unknown sites) and the graphic display. Generally, if the average priority value is quite high (approximately 10 and over) and no forbidden cubes or no occupied cubes with low (A<1 ) priority are present the test molecule will be formed rapidly by HLADH. If there are two atoms of test molecule In a boundary area (A< 2) formation is slow or does not occur. Figure 19 shows an example of a prediction table and a display of positions of the atoms of a test molecule in the cubic section model (co13). The test molecule is known not to be formed by HLADH reduction (the priority number is zero). Although no forbidden cubes are occupied one occupied cube has a very low priority value, 0.24. This suggests that the test molecule would not be expected to be produced by HLADH. The cubic section map for this molecule further showed that some carbons are near to forbidden cubes. It is usually better to check all visualisation methods when using ENZYME for prediction of reactivity, in practice it is found that the full prediction table gives the most accurate prediction. The cubic section model, co13 (Table 6) was used for prediction of the relative rates of formation for all 73 products in their different conformations listed in Table 3. Of these,
515
the more rigid were used in construction of the model but the less rigid (28) were not. For example. 3- and 4-alkyl cyclohexanols and heterocyclic bicycllc alcohols (Table 4) were not used in model construction. Because of the huge amount of data generated only twenty of the more interesting prediction results have been presented (Table 8). OH
layer 2 Si
IC H
9b
C I C -4-0—h-
layer 1
minimized structure
Type hoh.pre Substrate Original Priority: 0.00 Original State: forbidden ATOM ATOM # X
m - < »iiM
layer 0
H
layer -1
C 0 0 0 0 S O H C H
1 2 3 4 5 6 7 8 9 10
Y
Z
-1 1 0 0 0 0 -1 0 -2 -1 -1 -2 0 -2 0 -1 -2 1 1 -1 -2 0 0 1
CURRENT TOTAL AVERAGE #HITS PRIORITY PRIORITY 13 55 55 55 15 16 55 45 3 55
67.2 567.5 567.5 567.5 166.2 111.4 567.5 482.0 0.7 567.5
12.1 10.3 10.3 10.3 11.1 7.0 10.3 10.7 0.2 10.3
Total # atoms in molecule: 10 Average Priority of cubes over molecule: 9.3 Total # hits in cubes: 367 Average Priority for cubes aver all hits: 0.2 Number of unknown sites: 0 Number of forbidden sites: 0
Figure 19. Prediction table and part of displayed cubic section map for a test stoicture. The model col3. Only the forbidden cubes close to product atoms have been assigned.
516 Table 8. The prediction results for twent]^ test molecules using model co13. P=prlorlty A=average forbidden Test molecules Unk/Low 1 (expt) priority cube priority cube OH
.
^
,1,
10.1
OH
0
9.1
OK
0
9.1
OB
0
9.8
OE
0.05
8.5
OaM
0
7.6
OR.OaR
-2bF-'«=unk
0
7.6
0F,-2F
-2bA-i=unk
8.0 11.4
0A.-2A
0.9 OH OH
OH
OH
2.
(;V'^(46)
OH
OH
OH
3.
^
,S4, 0 52.67 OH
9.2
2E-1
0.03
10.0
2E
0.97
10.8
0 OH
\ n
517 [ T e s t molecules
P=priority (expt)
1 forbidden cube
A=average priority
Unk/Low 1 j priority cube
65.5
9.5
1A
2bA=unk
0
8.3
IF
2bF=unk
0
8.4
3E-2
2E-^
1.6
8.7
14.8
11.2
0
9.1
OH OH
^
3B"^=unk
OH
2F
2E
OH
1.2 0.02
120
10.6 1
8.6
1
2uE
2M=^unk
8.1 (28.8)
Ph
1
_''* AH
0
6.2
2F,3bF,3bL,2R
27.5
6.6(15.9)
5 cubes unk
0
6.5
5 cubes
518 A=average priority
P=priority (expt)
Test molecules
forbidden cube
, Unk/Low 1 priority cube
OH
32.6
13.9
0
9.3
12.4
10.9
0
9.3
2E-1
9.5
OH, OM
OH OH
'
OH OH
2F
y w '"' 1
1.0
1
^ OH
0
8.5
OR
0
8.9
-IB,-2A
0
8.5
-1E.2F
^ OH OH
9.
k J (62) OH
28.6 OH
1 -^J^
21.0
11.2 10.2
OK
519 [ Test molecules
1
A=average priority
P=priority (expt)
1
forbidden cube
lOnk/Low 1 priority cube
OH
J^
10.
(58) OH 1.3
7.8
0.06
8.5
3D"^
31, 3J=unk
OH
wax
3J, 2N=unk
(18)
ft
OH
3.0
10.6
0
7.7
3E
.0
1
10.1
1 laQ, laP, laO
0
1
6.7
laO, laP, laN
0
5.6
IF. OF
0
10.0
OA,OB, 1A
0
7.2
OA, OB
0
9.1
OF, OE, OL
(16)
12- k X j H
^
^ OH
1 ^^i
520 Test molecules
P=priority (expt)
00
A=average priority
forbidden cube
0
7.0
1L.0F.-1E
0
7.5
OA,-IB
0
5.2
IL.OF.OE.OB
0
5.7
OA, 08, OE
Unk/Low
1
priority cube
OH
06 06
r- A„'"' A>i,H
23.0
9.8(10.5)
3.0
9.2
U
0
9.4
U , OH
0
8.2
OP,OK
33.6
7.3(12.8)
-1H=unk
4.1
7.8(8.1)
-1 K=unk
0
9.3
U , IE-1
0
8.7
-IB-1
OH
A™ A^H MI
JO="
AV"
(30)
^
^
1
A:>OH
1
521 1 Test molecules
[
P=prJority (expt)
[
A=average [ priority
forbidden cube
rUnk/Low priority cube
r ^^''^ H O ^
1.5
8.5
1E'"'«unk
^
1.0
9.3
1B''*=unk
H
U
0
10.1
OH
0
8.9
OK, OP
13.6
9.7
IP
1.1
10.4
10
^
^ OH
17.
(42)
OH
OH
OH
L.
^
,44, OH
®
94.9
7.3(14.6)
1Q
5.1
9.9
1N.2E
522 Test molecules
A=average priority
forbidden cube
Unk/Low priority cube
(40)
19.
20.
P=priorJty (expt)
6.2
9.2
OH, U
1.3
9.6
OP, OH, 1H
9.7
IK, OK
9.4
0K.2K, U
x:b^
HO'
(70)
CO
91.3
11.5
laP, laQ
0.8
7.6
3E
81.6
7.2
OaO, laN
16.3
11.2
HOT'
xx>
Ha
For 2-methyl cyclohexanone (1), the first case in Table 8, the average priority value allows one to easily predict the main product of the reaction as the (IS, 2S) isomer, although one atom of the substrate occupies a low priority cube (OH).
The
enantiomeric (1R, 2R) product may be formed in small quantities, but formation of the other diastereoisomer (IS, 2R) and its enantiomer (1R, 2S) are clearly less likely due
523 to occupation of forbidden cubes. Our results agree with Jones prediction results for 2alkyl cyclohexanols (Figure 11). For test molecules cited In entries 2-4, 9, and 10 the assigned priorities are more difficult to compare with experiment because different reaction conditions were used in measuring their relative rates of formation. In entries 2 and 4 although many cubes of unknown priority are found, the calculated average priority values quite cleariy predict the relative proportions of products. For entry 10 cis/trans isomer ratios cannot be predicted. Only for 4-methyl cyclohexanols (entry 9) do the priority values predict cis/trans isomer ratios In agreement with experiment. The model (co13) accurately predicts preference for product formation from heterocyclic substrates with alkyl substitution. Good examples are entries 5, 7, and 8. If a phenyl group is appended to a heterocyclic ring predictions are less accurate because of the number of cubes of undefined priority occupied by this residue (entry 6). For bicycllc molecules (entries 11,12 and 13) the model accurately predicts which isomers which are not formed. Removal of bicyclo[3.2.1]octan-2-ol, entry 15, from model 0013 renders some cubes in layer-1 undefined priority, thus the priority values calculated for this case do not accurately predict exo/endo isomers. For bicyclo[2.2.1) heptan-2-ol, entry 14, priority values do accurately predict exo/endo isomer > 1 . Bicyclo[3.2.1]octan-2-one may effect the orientation of some amino acid side chains and the coenzyme which allows it to react with HLADH. The stereochemical course of reductions of some cage-shaped molecules (entries 16-19) is not well predicted. For entries 16 and 19 the products which are not formed (priority value is 0) occupy many low priority cubes. The major product with priority value 91.3 in entry 20 (new substrate) has a higher average priority value compared to other conformations but two atoms are in forbidden cubes. Based on this it can be assumed that these cubes could be allowed for some substrates instead of forbidden as shown in model co13. Models in which the origin was moved to the center of the center cube, the cube size was reduced to 0.65 A and hydrogens were added to the compounds used to construct the model and to the test compounds were next examined with 15 substrates to determine if improvements were achieved by these model modifications (Table 6, 9 and 10). Table 9 shows the predictions made by models of 1.3A and 0.65A cube size with the origin at the corner of the center cube with and without added hydrogens. Table 10 shows results obtained using the same models constructed with the origin in the center of the center cube. The average priority values of cubes over the substrates as well as the number of unknown sites and forbidden sites obtained for each substrate from each model were compared with experimentally derived priority values.
524 Table 9.
Prediction of acceptance by HLADH using cubic section models with 1.3A
or 0.65A cubes with origin at the corner of center cube, with and without added hydrogens. Average prority value, unknown and forbidden sites. Test molecule OH
P(expt)
32.6
co13
CO065
13.9,0,0 11.5,0,0
0013*^ C O 0 6 5 * co13h i 5o065h
14.4.0.0 11.7,0.0
11.4,0.0
10.5,0,0
9.2.0,1
8.0.0,0
7.9,0,3
11.0,0,0 11.1.0.0
11.1.0,0
11.2,1.0
9.2,0,0
9.9,0.0
7.5.0.0
10.6.0.0 10.3,1.0
10.2,1.0
9.3.5,0
7.5.0.1
7.2,0,3
8.0.0,2
7.2.0.7
10.1.0,3 7.3,0,4
6.9.0,8
5.5.0,14
OH OH 0
n
12.4
9.3.0,1
9.5,0,1
10.9.0,0 11.1.0.0
9.4,0.1
OH 0
9.3.0.0
9.5.0.0
9.4,0,0
OX'"' OH
J^
10.6.0.0 10.4.1.0
7.5.0.3
0
7.7.0.1
0
10.1.0.3 7.6,0.4
0
6.7.0.3
7.5.0.3
6.6.0.3
7.4,0,3
5.9,0.7 ! 4.6,0,13
33.6
7.3.1.0
6.8.3.0
7.5.1.0
6.9.3.0
5.0.0,0
3.7,10.0
4.1
7.8.1.0
7.8.3.0
7.5,1.0
7.5.1.0
6.5,0.0
3.7,12.0
0
9.3.0,0
7.2,0,4
9.1.0.0
6.8.0.4
8.2.0.0
6.0,0,6
8.8.0,0
5.6,0.4
8.8.0.0
(30)
/Kk°"
AT 1
3.0
1
^
/k^H
0
5.3,0.4
8.4.0.0
6.2,0,0
1
525 P(expt^
Test molecule
co13
CO065
co13*
CO065*
co13h |:o065h 1
^=fV^ (41) 1—1 ^
9.7
11.0.0,0 16.6,0,0
0.3
8.1,0,0
11.0,0,0 17.1,0,0
9.5,0,0
10.2,3,0
7.8,0,0
10.2,0,0
6.5,5,0
12.4,0,1 5.5,0,3
9.5,0,0
4.1,0,7
8.9,0,0
9.6,0,0
7.0,0,5
n 6.7.2,0
6.3,2,0
^ 0
12.1,0,1 5.8,0,3
^
1
*
_ X)H
1
0
1 9.0.0,0
9.2,0,2
9.1,0,3
J
In the first entry of Tables 9 and 10 predictions agree with the original model (co13), except for the enantiomer of the minor isomer (priority 0). For this compound ENZYME gives one or more forbidden sites for models coded co13h, co065h, ce065, ce065* and ce065h. In models co065h, ce065, ce065* and ce065h (Table 6) the priority values obtained do not accurately predict isomer preference. Entry 2 In Tables 9 and 10 is a good example of a case in which priority numbers accurately predict the outcome of reduction. For the bicyclic substrate (entry 3, Tables 9 & 10) the interesting result is that models with a 0.65 A cube size give many forbidden sites for endo products. In the case of cube file, eel 3 the same result was found. Many forbidden sites also are given for the enantiomer with a priority number of 0 in entry 4. We conclude that there are no significant differences between the predictive value of cubic section models wherein the origin is at the center or the corner of the center cube. Reducing the cube size from 1 .sA to 0.65A allows more accurate predictions to be made because positions of atoms in test compounds are more accurately defined. The presence or absence (coded with *) of a hydrogen on oxygen does not significantly Influence predictive value of the models. Thus, model col 3 can confidently be used to construct the shape of the substrate binding domain for HLADH and is probably the best model format for other dehydrogenases.
526 Table 10. Prediction of acceptance by HLADH using cubic section models with 1.3A or 0.65A cube sizes with the origin at center of the center cube, with and without added hydrogens. Average priority values, unknown and forbidden sites. P(expt) co13 C O 0 6 5 C 0 1 3 * C O 0 6 5 * co13h )o065h Test molecule OH
6."' 32.6
11.2,0,0 10.8,0.0
11.3.0,0 10.9.0,0
11.1.0,0
10.9,1.0
9.6,0.1
9.6,0,1
9.5.0.2
10.8.0,2
OH OH 0
12.4 '
10.4,0.1
10.4.0.1
10.5.0.0 10.8,0.0
10.5,0,0 10.9,0.0
10.9,0.0
11.2,0,0
9.7.0.0
9.6.0.0
9.5,0,0
9.4,0,3
11.5.0.0 10.1,1.0
10.2.2,0
10.1.2,0
OH OH 0
9.1.0.1
9.0.0.1
ccrH
"
OH
^
3.0
OH
11.4.0.0 10.6,1.0
0
7.7,0,3
7.9.0.3
7.5,0.3
7.6,0,3
8.5.0,5
7.1.0,8
0
7.2.0.4
5.4.0.4
6.9.0,4
5.0.0.4
8.7,0,5
5.5,0.11
0
7.2,0.3
5.4.0.3
6.9,0,3
5.0.0.3
8.1,0,5
7.5,0,9
33.6
8.2.0.0
6.2.1.0
8.0,0.0
5.8.1.0
6.3.3.0
7.2.6.0
4.1
8.5.0.0
6.6.2.0
8.3.0.0
6.2.2,0
7.3.3.0
7.5.6,0
0
9.6,0.2
7.0.1.1
9.5.0.2 ' 6.7,0.2
9.3,0,0
5.6,0.3
0
9.1,0.2
5.3.1,1
9.0.0.2
4.8.0.2
10.0.0,0
5.4.0,0
kr""' A>™ 1 1
\
1
h j^H
527
PrexDt^
Test molecule
co13
CO065
C013*
CO065* c o 1 3 h
bo065h
^^fY <*^) •-SOH
1—1
-S;
9.7
10.6.0,0 6.4.3.0
0.3
8.4.0.0
0
0
10.6.0,0 6.1.3.0
8.3.0.0
7.3,5,0
6.0.3.0
8.3.0.0
5.6.3.0
8.2.0,0
4.9,5,0
6.8.0,1
6.1.0.2
6.5.0.1
5.8.0.2
8.2.0,0
5.2,0.5
8.6.0.1
8.1,0,2
8.5,0.1
7.9.0.2
9.1,0,0
6.2.0.6
^
^
1^ 6.6.8.
Comparison of Present Models with X-Ray Crystallographic
Structure The X-ray crystallographic structure of the ternary complex, HLADH-NADH-dlmethyl sulfoxide, DIVISO, has been determined to 2.9 A.34 The crystallographic co-ordinates available from the Brookhaven protein data bank were entered into MACROMODEL and the amino acid residues surrounding the active site identified. Using MACROMODEL and ENZYME the substrate binding domain of HLADH was mapped as a cubic section model. The model obtained from this approach were compared with model col 3 obtained by substrate surrogate overlay. In the comparative study X-ray crystallographic data extending 15 A around the catalytic zinc atom was employed. Using MACROMODEL the sulphur of DMSO in the X-ray structure of the HLADH-NADH-DMSO complex was changed to a carbon reformulating this component as a 2-propanoxy anion with the carbon-oxyanion bond along the previous S-O axis and the carbon-hydrogen bond along to sulphur-lone pair axis. This alcohol structure positioned so that the oxyanion was located 2.2 A from the zinc. Distances between the substrate atoms and the HLADH-NADH residue atoms were determined using MACROMODEL. Table 11 shows the distances between the oxyanion (1), central carbon (1) and the neighbouring HLADH-NADH atoms within 4 A In this complex compared to similar distances for the HLADH-NADH-DMSO and
528 HLADH-NAD-p-bromobenzyl alcohol complexes. The locations of oxygen (1) and carbon (1) In the constructed 2-propanoxy anion-NADH-HLADH complex compared well with analogous distances in the other complexes. The distance from oxyanion (1) to the side chain atoms of HLADH differ only 0-0.5 A between the 2-propanoxyHLADH-NADH and DMSO-HLADH-NADH complexes and 0-0.6 A between the 2propanoxy-HLADH-NADH and PBBA-HLADH-NADH complexes. The location of
Table 11.
Interactions of three ternary complexes of HLADH and NADH. For the pbromobenzyl alcohol complex both observed and productive distances have been given 36, HIadh-Nadh ours HIadh-Nad1 -Dmso(A) Pbba(A) atom residue atom (A) 01
Zn Cys-46 Ser-48 His-67
Cys-174 CoE
C1
Zn Ser-48 His-67
SG OG CB CE-1 CD-2 NE-2 SG C4N C5N C6N
2.2 3.7 2.7
2.2
2.2 (2.2P)
3.6
3.4 (3.5p)
3.7 3.3 3.2
3.2 3.7 3.9
3.3 (3.4p) 3.8 (3.8p)
2.6 3.4
3.1
3.2 (3.2p)
3.6 3.0 3.2 3.8 2.9 3.3
3.5 (3.5p) 3.6 (3.5p) 3.4 (3.5p) 3.9(3.8p) 3.0 (3.1p) 3.4 (3.7p)
3.8 5.0 4.4
3.4
4.9
3.6 (3.6p)
3.6 OG CB CE-1 NE-2
'
3.7
CD-2 Phe-93
CE-2
3.5
4.2
Leu-141
CD-I
3.5
4.3
CZ
3.5
4.1
3.5 3.1
(3.5p)
Cys-174
SG
3.4
(3.6p)
CoE
C3N
3.2
(3.6p)
C4N C5N
5.1
2.4
(3.4p)
3.2
(3.7p)
1
529 carbon (1) differs 0.6-0.8 A In the case of DMSO-HLADH-NADH (except CN4 of NADH where a 2.7 A difference was obtained) and 0.1-1.3 A in the case of the PBBAHLADH-NADH complex from 2-propanoxy-HLADH-NADH complex. The embedded 2-propanoxy group the HLADH-NADH-2-propanoxy complex was oriented as previously described substrate surrogates using MACROMODEL Carbon (1),a-hydrogen and oxygen (1) of the 2-propanoxy anion were used to align the complex, other atoms of this product surrogate were deleted. The MACROMODEL. data file thus created was transferred to ENZYME using MMFORMAT. The complex was aligned as previously described for substrate surrogates and a cubic section model was created with a priority value of zero for all occupied cubes. The coordinates of cubes and atom types of cubes in this model were obtained by using PREDICT-and PRINT CUBEFILE-opWons. It contains 24 layers from-13 to 10. A printed version is available from the authors. We next determined the location in the cubic sector model of those residues lining the substrate binding domain of HLADH. Using the ANALYZE and DISPLAY modes of MACROMODEL (FINDNAME RESIDUE -options, MOLECULE, SET 1 and MONO -options) the residues near the active site pocket were identified and marked. Each marked residue (e.g. Phe-93) was separated from other residues using the DELETE ATOM / BOND command. Each separated residue was connected to the surrogate product to create a rigid complex as needed in the alignment process of ENZYME. Each of the files created by these operations was stored individually. The following residue files were formed: His-67, Cys-174, Cys-46, Leu-57, Ser-48, Phe-93, His-51, Leu-141, Met-306. Phe-110. lle-318, Pro-295, Val-294, Thr-94, Leu-92, Pro-91, Pro-95, Asp-49 and Val-52. These residue files were transferred to ENZYME, using MMFORMAT. The DISPLAY CUBEFILE option was used to determine the position of each residue within the cubic section model generated by ENZYME (shown as white circles each occupied cube). The co-ordinates of atoms in each residue were obtained from this process by creation of individual files for each residue and each of these was converted to a cubic section model. Addition of the residue cubic section models gave a cubic sector model of the substrate binding domain consisting only of the residues lining the this pocket. Model 0013 and the model constructed with all hydrogens in substrate surrogates (co13h) were compared with the cubic section model derived from display of the cubes occupied by residues of HLADH lining the substrate binding domain. Using
530 COMPARE the X-ray based model was overlaid with the substrate surrogate derived models.
Forbidden cubes in c o 1 3 . c o 1 3 h and the X-ray derived model which
overlaid were identified and displayed (Figure 20). The co-ordinates of each cube and atom types in each cube were also identified. This allowed location of the residues lining the substrate binding domain that interfered with substrate binding. Only layers between -3 to 4 have significant overlap between the models. We commenced analysis of layers at layer -3, then proceeded to layer -2 and so on. The coenzyme, NADH was located in the front layers. The forbidden cubes in layers -3, -2 and -1 are close to the coenzyme and residues Phe-93, lle-318, Cys-174 and His-67. Residues Leu-309, Val-309 and Thr-94 with attached hydrogens may effect layer 0. The coenzyme and Phe-93 are the closest to the cubes -1H, -1K and -10 In model c o l 3 . For some bridged substrates (30, 31) these cubes are allowed in this model. In layer 0 the residues Leu-309, lle-318 and Val-294 affect the cubes above OM to OR and may be In the boundary area. Residues Phe-93, lle-318 and possibly Hls-67 affect the forbidden cubes on the right side of model co13. Val-294, Ser-48 and coenzyme are the residues nearest to the forbidden left edge. According to the Xray derived cubic section model the left side of the substrate binding domain (in layers 1-4) Is more open because Val-294 and Leu-309 can change orientation. In layer 1 Phe-93, Thr-94, His-67 and lle-318 affect the forbidden right side and lle-318 affects the cubes above ON to OQ in model co13. Ser-48 and His-51 are quite close to the left edge of co13. This edge is assumed to be the boundary area. It is forbidden for some substrates and allowed for others. In layer 2 the right edge of co13 Is affected by Hls-67, Phe-93, Leu-116, Leu-141, lle-318 and Phe-110. The Van der Waals interactions from other layers (1 and 3) have an especially important role in layer 2 definition. His-51 and Ser-48 may affect the left edge, an area that seems to be allowed for substrates with phenyl groups. The upper part of the left side of co13 is the most open with Val-294, Leu-309 and Pro-295 as the nearest residues. The same residues affect layers 2 and 3. Whereas residues Leu-57 and Phe-140 affect layer 4. The cubes in the upper edge of the left side of layers 2 and 3 are defined from larger substrate surrogates. They may affect orientations of the side chains of Val-294 and Leu-309. Leu-116 and Met-306 have also been suggested to change their side chain conformations with more bulky substrates (38) however, Met-306 Is quite deep In layers 3 and 4 In present X-ray based model. Comparison of the shapes of the substrate binding domain derived from these two approaches provides a good view of the domain.
531 Jones manually derived positions of amino acid residues in the apoenzyme structure and some ternary complexes published before 1977. Figure 21 shows a comparison between the present results with the earlier definition. The boundary region of model (0013) has also been assigned in the Figure. Ser-48, Phe-93 and lle-318 are in the same location while Leu-57 is in layers 3, 4 and 5 In our X-ray derived model. Using the present method positions of residues were positioned more accurately.
(H)
(C)
(N)
(N)
nil [TJ G
(Ip) CoE (C,H)
(0)
(Ip)
CoE
CoE
CoE
A
0
P
Q
Tn
H
1(H) J
K
L
B
CoE D C CoE^)
E
F
uE
uF
uA(C •uB(C) uC(K) uD |coE CoE CoE
(C)
(S) C174 (H) (C) (H) C174 a? A
(C)
(H) C-46
layer -3 (C)
CI 74 stoiTi type (C) and the amino acid residue, Cys-174 forbidden allowed for some 3-alkylcyclohexanols or heterocyclic bicyclic alcohols, usually forbidden allowed for 4-alkylcyclohexanols, usually unknown Figure 20. The overlaid layers with X-ray based model and present models oo13 (upper) and oo13h (lower).
532
(0) 1-318
(0)
1-318 (H) (H) CoE 1-318 (C)
(H)
(0)
(H) CoE
F M ^ ^ G (C) CoE BCoB
[uA
(C) CoE CoE
(N) CoE
acog adoB J
"(C) '(C) CoE CoE C D B
uB
(H)
(H)
o u C ^ uD
1'1 K 1
E
uE
""(Hi P-93J uF
(H)
|p-93 (H) CoE (0)
(H)
(C)
(H)
C-46 (S)
C-46 (H) (C) C-46 C-46
layer - 2
(H)
(H)
533
RcJo
pcoTl
1-318 (C) h-318 1 (C)
pi (H) CoE
G
[0 1 0.02 J H 1 1 34
C
A
(Ip) CoE
(C) ! uA CoE
(0.1 P: qoE
TH
[N
NJB
K 1 L 4
p 55 ^ ^
uC ^ uD 45 11
uE
uF
(C,C) |P-93 (H) P-93
10 9 (H) H-67
(H)
(c)
(C)|
(N) H-67
(C) (C,H)i (N)
(c) 1 (H)
(0)1 H-67
C-46 C-46
layer -1 (C,H) 1-318
(0)
(C) 1-318 (C)
(H) CoE
[G A
(Ip) CoE
0 2 P 2^ ^ R 3 17 JHIC 1 9 J 6 K 3 2 4 4 16 D 55 E 3 B 2 c 2
(C) CoE
Ko.ip;
12
^ ^ u C
^ uD 1 uE
44 11
10 8
JP93| KC^ P-93
WPS
(H)
(H) H-67
(C) (C.H)
mi
1 I
(C)
1
(N) H-67
(N)
(C) (H) C-46 C-46
(0) |H-67
534
layer 0
535
layer 1
536
537
ric)
(H)
rw
M-306 M-306
(H)
P-110
P-295
p-no
(H)
(H) V-294
[ M ^ pT"
PTHT \Q
0
[R
1
L-116
H G 61'^ V-294 H-294 2 A "Z B 'A 61 11 31 (H)
uA
c
ZTD 1
•(d) P-93
A^yV.
uB
rTc)
u E ( H ^ ^ a (H) (C) L-141 (H)
1 28
H-51
1 28 , (H)
(0)
H-51 (N)j
S-48 (H)|
H-5l!
A-49
L-141
(Ip.O)
{H)| P-14(^
layer 3 (C)\
(C)
M-306 M-306
p-no (H)
(H) (H)
p-no
P-29S 1 (C) |L-n6
11 121 (H) V-294
IM
N
\ 0
R
^J»57
(C)
3 4
(H) H-51 (N) H-51
1
1.3 H 4 1
G 5J 4 61^ 61^ 7 1 25 ^ ^ ^ 4 V-294 H-294 4 A 7 B 13 C 5| D 8 E 4 1 121 46 17 16 1 1 8^ 0.6^ 19 10 2 2
H-51
^P
28^ 0,48
"loTJ uF 1 m
p-93
^1
(C) l " 2 16 ^ ^ 0.22 L-141 (H) 1 28 L-141 1 4
(H)
(H)
A-49
P-140
moi
538 IHTI
(H)
(H)
M-306
M-306
p-no
(H) L-116
(H) L-ne
[M
(H)
(H)
N
0
P
1
•
G
H
A
B (H
>oo?
uA
L-57 uB(C) L-57
L-57
lr-57
Q(C) L-ne K
D
"Wl
(H)
"mlflcT 1 L-n 6 1-116 L
F(H)
(H)
L-14l| | L - 1 4 1
^uD
(H)
• 1
(H) L-141 (C)
L-141
H-51
(C) L-141
(C)
(H)
(C,H)
(C,H)
H-51
H-51
P-140
L-141
(H)
(C) 1 ^^^ P-140
H-51 (H) V-52
layer 4 (H)
(H)
(H)
M-306
M-306
p-110
(H) L-116
(H) L-ne
\IA
(H)
(H)
r^
0
H
A
B 1 C 1 3.5 3.0 H,57 uB(C]
uA
L-57|
P
K
1
^ ^ ^^^ ^^^
L
F(H)
(H)
L-14l| L-141 uF
(H)
1 57K
L-141 (C)
(H)
(C)
L-141 L-141
H-51 (C)
(H)
1 L-116 L-116| [1-116
G
L-57
(HTI
(H)
H-51 H-51
(C.H)
(C,H)
P-140
L-141
(C)
(C)
H-51
p-140 (H) V-52
(H)
539 Jones model 309
[306
3od sial am
p!95
cW\ ^
294 294 294
layer 2
Ju r
mo
>^;
294
110
51
1''
^
51
1411 48
48
I 93
1141
48
HO]
48
110^
309
309
bisj
295
318 318
i94^H
294
layer 1
94
294 93 MM
k^
1
1
51
48
309
48
no^
bisl
309
318
309
3ji
COEI
iK
c^
S — < ,—1
• H ESil JS? mm w^m mm ^mm >S$^ layer 0
u
93
CoE
|H-5J
CoE
^
M
—<
MP
48 48
-^^^ . . • torDidden [ ^ BMhJ or limited
93
93
48 67
, „ „. allowed boundary \ ^ in some KX^ ^^^^«
94
^Mji"
[2*94^^
294
^.^.^^
Ur
67
^
1
forbidden
Figure 21. Comparison of Jones map of amino acid residues with present definition.
540 6.7.
CONCLUSIONS
The present computer-based substrate surrogate overlay method gave a refined cubic section model for the substrate binding cavity of HLADH. Allowed and forbidden regions were accurately defined using the origin in the corner of a cube and a cube size 1.3 A or 0.65A (model co13 and co065). Boundary regions (Figure 21) which can be either allowed or forbidden were defined. The average acceptance value in these regions was usually 2 or lower. Low average priority values for some cubes (Figure 16: OG, ON, OQ, OK, 1 G, 1 K, l u E , 2K, and 2E) are due to the inclusion of bulky substrate surrogates with low priority values in construction of the model. These are assumed to change the orientation of the side chains of amino acid residues lining the substrate binding domain. Reducing the cube size to 0.65A (co065) from l.sA (co13) primarily affects cubes OH, OK, ON, OQ, 1K, 1H, and IN in the 1.3A model by defining those parts of these boundary cubes that are forbidden or allowed. The cubic section models, co13 and co065, proved to be accurate in predicting acceptance of substrates and the stereochemical course of HLADH mediated reductions. The average priority value for all cubes, number of forbidden sites and number of low priority cubes occupied by atoms in a possible alcohol are used to estimate the acceptability of the corresponding ketone by HLADH. The prediction method developed can be applied to other enzymes (e.g. other dehydrogenases, esterases, or lipases) if kinetic and specificity data is available. Comparison of the X-ray based cubic section model with the substrate surrogate derived cubic section model gave a view of the amino acid residues around the substrate binding domain (Figure 21). Forbidden and boundary regions defined by the substrate surrogate overlay method are near the side chains amino acid residues. Substrate interactions with surface of the amino acid residues could be estimated by molecular mechanics (MACROMODEL). The close resemblance of the present models, developed with rather sophisticated computer techniques, to the original Jones model, developed with hand held stick models, is a testimony to the ingenuity of the Jones group. 6.8.
ACKNOWLEDGEMENTS The authors acknowledge the financial support of Suomen Kulttuurirahasto,
Neste Oy and Kemira Oy of Finland and the Natural Sciences and Engineering
541 Research Council of Canada. As well the authors thank Mr. L. Corey and Ms. Sharon Cyprik for development of the ENZYME and COMPARE programs and Clint Surry and Dr. All Mohammed for help in preparation of the manuscript. M.A. sincerely thanks Professor H. Krieger for his support and encouragement of her studies in Canada.
6.9. REFERENCES 1. E. L Smith, R. L Hill, I. R. Lehman, R. J. Lefkowitz, P. Handler & A. White, in: R. S. Laufer, E. Warren & D. Mclvor (Eds), Principles of Biochemistry: general aspects, R. P. Donnelley & Sons Company, 1983. 2. C.-H. Wong, Science, 244 (1989) 1145. 3. A. Akiyama, M. D. Bednarski, M. J. Kim, E. S. Simon, H. Waldmann & G. M. Whitesides, Chemtech, (October 1988) 627. 4. J. B. Jones, Tetrahedron, 42 (1986) 3351. 5. G. M. Whitesides & C.-H. Wong, Angewandte Chemie, 24 (1985) 617. 6. M. Schneider, Enzymes as Catalysts in Organic Synthesis, Reidel, Dordrecht,1986. 7. J. B. Jones, in: P. Dunnill, A. Wiseman and N. Blakeborough (Eds), Enzymes and Nonenzymic Catalysis, Horwood/Wiley, Chichester/New York,1980. 8. J. B. Jones, C. J. Sih & D. Perlman, Applications of Biochemical Systems in Organic Chemistry, John Wiley & Sons, inc., 1976. 9. J. Retey & J. A. Robinson, Stereospeciflcity in Organic Chemistry and Enzymology, Verlag Chemie, Weinhelm/Deerfield Beach, Florida/Basel,1982. 10. A. Fischli, in: R. Scheffold (Ed.), Modern Synthetic Methods, Vol. 2, SalleSauerlander, Frankfurt, 1980. 11. J. B. Jones, in: J. R. Morrison (Ed.), Asymmetric Synthesis, Vol. 5, Academic Press, New York, 1980. 12. P. Sonnet, Chemtech, (Fehruary 1988) 94. 13. Enzyme Nomenclature, Elsevier, Amsterdam, 1973.
542 14. K. Martinek & J. J. Semenov, Appl. Biochem., 3 (1981) 93. 15. H. Theorell, in: R. G. Thurman, T. Yonetani, J. R. Williamson & B. Chance (Eds.), Alcohol and Aldehyde Metabolizing Systems, Academic Press, Inc.,1974. 16. C.-l. Branden, H. Jornvall, H. EkIund & B. Furugen, in: P.D. Boyer (Ed.), The Enzymes, Vol. XI, Part A, Academic Press, New York, 1975. 17. H. EkIund & C.-l. Branden, in: T. G. Spiro (Ed.), Zinc Enzymes, John Wiley & Sons, Inc., 1983. 18. H. EkIund & C.-l. Branden, in: F. A. Jurnak and A. McPherson (Eds), Biological Macromolecules and Assembles, Vol. 3, John Wiley & Sons, Inc., 1983. 19. V. C. Sekhar & B. V. Blapp, Biochemistry, 27 (1988) 5082. 20. J. P. Klinman, CRC Critical Reviews in Biochemistry, 10 (1981) 39. 21. J. B. Jones, Enzyme Engineering, 6 (1982)107. 22. A. I. Irwin & J. B. Jones, J. Am. Chem. Soc, 98 (1976) 8476. 23. D. R. Dodds & J. B. Jones, J. Chem. Soc, Chem. Commun., (1982).1080; J. Am. Chem. Soc, 110(1988)577. 24. A. J. Irwin & J. B. Jones, J. Am. Chem. Soc. 99 (1976),1625. 25. I. J. Jacovac, G. Ng, K. P. Lok, & J. B. Jones, J. Chem. Soc, Chem. Commun., (1980) 515. 26. J. Davies & J. B. Jones, J. Am. Chem. Soc, 101 (1979) 5405. 27. M. A. Findeis & G. M. Whitesides, Ann. Rep. Med. Chem., 19 (1984) 263. 28. J. B. Jones & K. E. Taylor, Can. J. Chem., 54 (1976) 2969. 29. C.-H. Wong & G. M. Whitesides, J. Amer. Chem. Soc, 103 (1981) 4890. 30. W. H. Baricos, R. P. Chambers & N. Cohen. Anal. Lett., 9 (1976) 257. 31. A. R. Fersht, in: Freeman (Ed.), Enzyme Structure and Mechanism, New York, 1985.
543 32. R. W. Hay, Bio-inorganic Chemistry, Ellis Norwood, Halsted Press, 1989. 33. H. Ekiund, B. Nordstrom, E. Zeppezauer, G. Soderlund, I. Ohisson, T. Boiwe, B.-O. Soderberg, O. Tapia, C.-l. Branden, and A. Akerson, J. Mol. Biol., 102 (1976) 27. 34. H. Ekiund, J.-P. Samama, L Wallen, C.-l. Brand6n, A. Akeson & T. A. Jones, J. Mol. Biol., 146(1981)561. 35. H. Ekiund, J.-P. Samama & L Wallen, Biochem., 21 (1982) 4858. 36. H. Ekiund, B. V. Plapp, J.-P. Samama & C.-l. BrSnd^n, The J. of Biol. Chem., 257 (1982)14349. 37. E. Cedergren-Zeppezauer, J.-P. Samama & H. Ekiund, Biochem., 21 (1982) 4895. 38. E. Horjales, H. Ekiund & C.-l. Brarlden, J. Mol. Biol., 197 (1987) 685. 39. H. Jornvall, Eur. J. Biochem., 16 (1970) 25. 40. H. Ekiund, Biochem. Soc. Trans., 17 (1989) 293. 41. V. Prelog, Pure Appl. Chem., 9 (1964) 119. 42. A. J. Irwin & J. B. Jones, J. Am. Chem. Soc, 99 (1977) 556. 43. A. J. iHA^in & J. B. Jones, J. Am. Chem. Soc, 99 (1977) 1625. 44. E. Horjales & C.-l. Branden, J. Biol. Chem,, 260 (1985) 15445. 45. J. B. Jones & I. J. Jacovac, Can. J. Chem., 60 (1982) 19. 46. J. M. H. Graves, A. Clark & H. J. Ringold, Biochem., 4 (1965) 2655. 47. M. Nakazaki, H. Chlkamatsu, K. Naemura, Y. Sasaki & T. FujIjI, J. Chem. Soc, Chem. Commun., (1980) 626. 48. H. Duller & C.-l. Branden, Bioorg. Chem., 10 (1981) 1. 49. G. L Lemiere, T. A. Van Osselaer, J. A. Lepolvre & F. C. Alderweireldt, J. Chem. Soc, Perkin Trans II, (1982) 1123.
544 50. R. A. Johnson, in: W. S. Trahanovsky (Ed.), In Oxidation in Organic Chemistry, Part C. Academic Press, New York, 1978. 51. M. Nakazaki, H. Chikarnatsu, K. Naemura & M. Asao, J. Org. Chem., 45 (1980) 4432. 52. J. J. Willaert, G. L. Lemiere, L. A. Joris, J. A. Lepoivre & F. C. Alderweireldt, Bioorganic Chemistry, 16 (1988) 223. 53. J. B. Jones & T. Takemura, Can. J. Chem., 60 (1982) 2950. 54. J. A. Haslegrave & J. B. Jones, J. Am. Chem. Soc, 104 (1982) 4666. 55. T. Takemura & J. B. Jones, J. Org. Chem., 48 (1983) 791. 56. M. Nakazaki, H. Chikamatsu & Y. Sasaki, J. Org. Chem., 48 (1983) 2506. 57. M. Nakazaki, H. Ckikamatsu & Y. Sasaki, J. Org. Chem., 48 (1983) 4337. 58. M. Nakazaki, H. Chikamatsu, K. Naemura, T. Suzuki, M. Iwasaki, Y. Sasaki & T. Fujiji. J. Org. Chem., 46 (1981) 2726. 59. T. A. Van Osselaer, G. L Lemiere, J. A. Lepoivre & F. C. Alderweireldt, Bull. Soc. Chim. Belg., 89(1980)389. 60. T. A. Van Osselaer, G. L, Lemiere, J. A. Lepoivre & F.C. Alderweireldt, Bull. Soc. Chim. Belg., 89(1980) 133. 61. J. Van Luppen, J. A. Lepoivre, G. L. Lemiere & F. C. Alderweireldt, Heterocycles, 22 (1984) 749 62. A. R. Krawczyk & J. B. Jones, J. Org. Chem., 54 (1989)1795. 63. L K. P. Lam, I. A. Gair & J. B. Jones, J. Org. Chem, 53 (1988) 1611. 64. C.-S. Chen, Y. Fujinloto, G. Girdaukas & C. J. Sih, J. Am. Chem. Soc, 104 (1982) 7294.
545 6.10.
Appendices
Appendix 1 : MACROIVIODEL Description After connecting to the VAX and setting the correct sub directory (e.g. set default [site]), the program MACROMODEL is started by typing RMMOD. When the display is shown on the screen, active buttons are shown in green. The first mouse key is shown as the pick key. The second mouse key acts the same for ail buttons except for the DRAW button. The third mouse key will display the help screen for that button. The molecule on the screen will be drawn by using the DRAW button active. The first key draws connecting bonds to subsequent points whereas the second key restarts the drawing of bonds. The hydrogens are added by choosing the H ADD button three times. Non carbons are replaced by choosing the appropriate symbol and picking the atom to replace. Functional groups can also be appended in this manner. The structure is then minimized in energy by selecting the ENERGY mode (choose MM2 force field) and picking start.
Constraints are added by selecting the
CONSTRAINT submode and selecting the appropriate choice of constraint (CATM, CDIS, CBOND, CTOR) and choosing the atoms involved. To orient the structure, pick the ANALYZE mode and pick ALIGN X, ALIGN Y, ALIGN Z, ROTX, ROT / a n d ROTZ as needed. The aline buttons need two atoms picked after the command to align on the axis and the rotate buttons need a typed input on the number of degrees to rotate on that axis. The structure is stored by picking the WRITE button an supplying a filename (the extension defaults to .DAT) and give a blank line for sequence structure number. When finished, exit the program by picking the STOP button and confirming that you wish to exit. Answer "Y" to the question for deleting the log file.
546 Appendix 2:
ENZYME and COMPARE Documentation
To execute the ENZYME program, type RUN ENZYME and the graphics screen with a menu is displayed as in Figure 12. The choice of options are: READ CUBEFILE, READ DATA, ADD TO CUBE FILE, REMOVE FROM CUBEFILE, ALIGNMENT, ROTATE ON X AXIS, ROTATE ON Y AXIS, ROTATE ON Z AXIS, PREDICT PRIORITY FOR MOLECULE, DISPLAY CUBEFILE, ROTATE ABOUT BOND, PRINT CUBEFILE, SAVE CUBEFILE, EXIT, CHANGE CUBE SIZE, CHANGE ORIGIN IN CUBE, and DISPLAY CUBE WITH DIFFERENT PRECISION. The desired option is chosen by depressing a mouse button while pointing the cursor at the option and the exact operation to be performed is dependent on which of three buttons was pressed. 1. Read cube file {READ CF): This option asks for the cube file to read; the extension .cf will be added to the file name. 2. Read data (READ DATA): This option reads in the data generated by MACROMODEL program (the extension must be '. dat' and the file must be in ASCII format which is made by running MMFORMATor\ the file made by MACROMODEL and supplying the filename) and asks for the necessary information to determine its priority. The first button will direct the program to follow this section by the alignment section; the second button directs the program to return to the menu. 3. Add to cube file {ADD TO CF): This option adds the molecule to the cube file; It first checks to see if the molecule has already been aliened. 4. Remove from cube file {REMOVE CF): This option removes the priority from the cubes that are occupied by the molecule. It does not check the cube file for the presence of the molecule since it is impossible to confirm the previous addition of the molecule. The molecule must have the same priority ranking as it had when it was added. 5. Alignment {ALIGNMENT): The option aligns the molecule in the reference axes with one of the atoms on the origin with a relative co-ordinate of (0, 0, 0). It will ask for the atom to be placed on the origin, the atom to be on an axis which is chosen by one of the buttons, and the atom to be placed in a plane which is chosen by one on the buttons. When choosing this section with the second button, the atom chosen will be translated to the origin and the alignment of the rest molecule is not done. 6. Rotate on x axis {ROTATE ON X), rotate on y axis {ROTATE ON Y), rotate on z axis {ROTATE ON Z): These options will rotate the molecule on the specified axis by an
547 amount which is typed in. Depression of the second button will rotate the molecule on the axis by 180**. All rotations are positive in the right handed co-ordinate system. 7. Predict priority for molecule (PREDICT): This option determines the priority of the molecule based on the information in the cube file. The second button will write the information, and how each atom fits, in a file with the same name as the molecule but with the extension '.pre*. 8. Display cube file (DISPLAY CF): This option displays the cube file by displaying each layer of the cube file starting from the near side. It will either display the cube file with 3 colors or 6 colors depending on the value shown for the last option. The displayed layers will be limited (from -5 to +10 inclusive) if the first button is pressed and will be unlimited if the second button is pressed. If a molecule shown on the screen, then circles within the cubes are drawn to indicate which cubes the molecule occupies. 9. Rotate about bonds (ROTATE BOND): This option rotates a group of atoms the specified bond and returns the orientation with the best fit for the cube file; it does not add the molecule to the cube file. 10. Print cube file (PRINT CF): This creates a file which contains the cube coordinates and the average priority for the cube. It also includes the number of atoms in the cube. 11. Save cube file (SAVE CF): This saves the cube file. 12. Exit (EXIT): This option also saves the cube file if it has been changed and exits the program. 13. Change cube size (CF number): This option allows the size of the cube to be changed. It does not recalculate the cube file to reflect a change in size of the cube so a different cube file should always be used with each cube size. If you wish a size different from the default, specify the change in size before doing any operations on the cube file so the cube file will not contain garbage. 14. Change origin in cube (CORNER or CENTER): This option moves the origin of the axes to the corner of the cube or the center of the cube. The place on the origin is reflected by location of the origin, specify the change before doing any operations on the cube file. 15. Display cube with different precision (DISCOL number): This option determines if the cube file Is displayed In 3 or 6 colors with different ranges.
548 To Start to execution of COMPARE, type RUN COMPARE after exiting ENZYME program. Then choose option READ CUBEFILE and give the name of first cube file and also the name of second cube file. After it choose option DISPLAY CUBEFILE and it gives the overlaid picture of these cube files. The overlaid cubes can be seen with different colors: forbidden in first cube file (black), forbidden in both cube files (red), conflict in first cube file (blue), conflict in second cube file (yellow), and allowed in both cube files (green). In the end of the program it gives the number of conflicts in both cube files.
479
Modelling the Substrate Binding Domain of Horse Liver Alcohol Dehydrogenase, HLADH, by Computer Aided Substrate Overlay Maija Aksela and A.C. Oelilschlager
6.1.
INTRODUCTION
Enzymes are catalytically active proteins that are involved in every in vivo transformation. They enhance the rates of biochemical reactions by 10^ to 10^2 by reduction of the free energy of activation J Two distinctive properties of enzymes are their high substrate specificity and the narrow range of conditions under which they are effective. They usually catalyze one reaction of a few substrates. Activities are dependent on pH, temperature, the presence of cofactors, as well as concentrations of substrates and products. Enzymes perform specific reactions because they possess cavities in which substrates are oriented while they are transformed (Figure 1). This process Involves interaction of the substrate with amino acids of the enzyme. Enzyme-substrate complexes have been studied by kinetic analysis, chemical modification, inhibition of enzymes by specific compounds that Interact with active sites, detection of characteristic spectral absorption bands during reaction of enzymes with substrates, and X-ray crystallographic analysis of enzymes combined with compounds which are in similar structure to the natural substrates. The Interaction between enzymes and substrates has been analyzed by the concepts of "lock-andkey" and "Induced fit". The former presumes that the substrate surface must fit the enzyme surface like a key In a lock, while the latter refined theory assumes that binding of the substrate induces conformational changes in the enzyme to provide a better fit. 6.2.
Enzymes as Catalysts in Organic Synthesis
The potential of enzymes as catalysts in asymmetric synthesis has been recognised for many years.2-12 Rate acceleration and stereoselectivity, together with techniques for the low-cost production and the rational alteration of their properties, make enzymes attractive as chiral catalysts in organic synthesis. Enzyme-catalyzed reactions have been categorised into six main groups''^ as shown In Table 1. Three of them, oxldoreductases, hydrolases, and lyases have been found useful In organic synthesis.
480 About 50 of the 200 enzymes produced industrially have been shown to be important to synthesis.I"* Active site
© Enzyme
Substrate
Enzyme-Substrate Complex
Product
E-Product
Figure 1. Schematic diagram of an enzyme-catalyzed reaction. Table 1. Enzyme classification. Class
Action
Oxido-reductase
Oxidation-reduction reactions.
Transferase
Transfer of a functional group (e.g. amino, methyl) from one substrate to another.
Hydrolase
Bond cleavage by addition of water.
Lyase
Reactions involving additions to double bonds or cleavages with formation of double bonds.
Isomerase
Ligase
Racemization of optical or geometric isomers and certain intramolecular oxidation- reductions. Formation
of
new
triphosphate (ATP).
bonds, cleavage
of
adenosine
481 The chemo-, regio- and enantloselectivlty of enzymes make them Ideal asymmetric catalysts. Most Important Is the differential recognition of diastereotopic groups of chlral and prochlral substrates. The mild conditions under which most enzymes operate minimize Isomerizatlon, epimerlzation and racemization associated with many other chemical processes.^.Q Maturation of the petro-chemlcal Industry, environmental pressures for "clean chemistry" and the explosive development of biotechnology have increased interest in the application of enzymatic processes to organic synthesls.3 Enzymatic processes play an increasing role In the generation of chlral pharmaceutical Intermediates, watersoluble materials and Copolymers. One problem In the development of enzymatic reactions for organic synthesis Is the prediction of the stereochemistry of reaction. Reliable models for prediction of stereochemistry are needed to broaden the application of enzymes to organic synthesis. 6.3.
Horse Liver Alcohol Dehydrogenase as a Catalyst
Horse liver alcohol dehydrogenase, HLADH, (also abbreviated as ADH or LADH) is the most extensively studied oxido-reductase. It plays a central role in ethanol metabolism and has been one of the main tools for understanding the mechanism of this process.'iS it was crystallized from horse liver In 1948 by Bonnlchen and Wassen and Is commercially available. Three Isozymes EE, ES and SS are formed by dimeric combination of two different, E or S (E "ethanol-active" and S "steroid active"), protein chains.''6 The EE- Isozyme of HLADH has been used in organic synthesis. HLADH Is nicotinamide coenzyme {NAD+/NADH) dependent and catalyzes the redox equilibrium between a large number of alcohols and ketones or aldehydes (Figure 2). The equilibrium is overwhelmingly in favour of the reduction reaction.^ Phosphate analogs of the coenzymes, NADP+ and NADP may also serve as coenzymes in very limited situations. The oxidation-reduction takes place through a ternary complex in which the substrate and coenzyme are simultaneously bound In the active site of enzyme. A zinc Ion at the active site binds the substrate and facilitates hydrogen transfer by acting as a Lewis acld.17 it has been assumed that the alcohol forms a zinc bound alkoxide during the reaction.is His-51 has been suggested to be important In transfer of a proton between the active site and surrounding solvent.''® Which amino acids are
482
9
HH..
R
H.N'Y^ OII '^ i A- --fNADH
NAD-"
NH2 HO
OH
^ O
O
^
OH
OH
N ^T^ll
^ 3 HO
f OH
Figure 2. Schematic view of the oxido-reduction catalyzed by HLADH. responsible for the observed pH dependencies of coenzyme association and dissociation as well as substrate binding and hydrogen transfer steps are not yet certain.19 The oxidation has been suggested to occur by an ordered mechanism in which the productive substrate binding site is formed after coenzyme binding. Dissociation of the enzyme-NADH is the rate limiting step.20 OH
f
CH3OH R
HO'
RCH2OH
R'
bnoH R'
d 6T Q5^
YADH HLADH Steroid alcohol dehydrogenase
Figure 3. Structural
specificity
of
HLADH
compared
with
dehydrogenase, YADH and steroid alcohol dehydrogenase.
yeast
alcohol
483 Over one hundred compounds have been examined as substrates for HLADH. It has a broad structural specificity and is well suited for organic synthesis. The enzymes reacts with acyclic, mono-, bi-, tri- and tetracyclic (steroidal) substrates. Figure 3 shows a comparison of the substrate specificity of common dehydrogenases.^^ HLADH has a wider use than yeast alcohol or steroid alcohol dehydrogenases. It operates on most substrates with high enantiomeric specificity. Examples in which HLADH exhibits superior selectivity include preparation of stereoisomers of bridged bicyclic
(±)
O unreactive enantiomer
83% e.e.
2.
100% e.e. H
H
(±) P
^OH
3. XH2CH2OH
'CH2CH2OH
(±)
4.
97% e.e.
J 10'''^ HO-^
L ^OH ^OH (±)
O
^
J^ J a'^^^ O'^'^X)-^ 100% e.e. OH
^ ^ unreactive enantiomer
"^ " ^
macrolide and polyether antibiotics
OH
5. (±)
100% e.e.
Figure 4. Examples of HLADH-catalyzed reactions.
100% e.e.
484 compounds (entry 1 in Figure 4).22 Stereospecific reduction of highly symmetrical diketones is also facile (entry 2, Figure 4).23 it is also possible to combine several different kinds of specificity to achieve in a single step a transformation that usually requires several reactions (entry 3, Figure 4).24 Chiral lactones, which are useful starting materials for many natural products, can be formed by stereospecific oxidations of mesodlols (entry 4, Figure 4).25 Even thioketones are reduced stereospecifically (entry 5, Figure 4).26 Reactions with HLADH typically occur at temperatures between 4°C and 25°C and in the pH range of 5 to 10. For catalysis of a reduction the optimum pH is ~7 while for the reverse oxidation it is -8.^ Reaction times vary from a few hours in the most favourable substrates and 2-3 weeks for the slowest. The disadvantage of HLADH has been the high cost of coenzymes.
Fortunately, several recycling methods are available that
allow reduction of substrates at the research scale (up to 1 kg of substrate).27-30 Por example, the ethanol-coupled method has been used for reduction and flavin mononucleotide (FMN) recycling for oxidation. 6.4.
X-Ray Crystallographic Studies of HLADH
Understanding of protein structure and function has been greatly enhanced by X-ray crystallography. At low resolution (4-6 Angstrom), the electron density map reveals the folding of the polypeptide chain, but few other structural details. At 3.0 A , it is possible, in favourable cases, to resolve amino acid chains, while at 2.5 A the positions of atoms may often be given with an accuracy of ± 0.4 A. In order to locate atoms to 0.2 A, a resolution of about 1.9 A and very well ordered crystals are necessary.3i.32 Three different crystal forms of HLADH have been studied crystallographically: orthorhombic (C222) crystals of the apoenzyme (native enzyme), triclinic (PI) or monoclinic (P2-|) crystals of the holoenzyme (apoenzyme with its coenzyme) and of ternary complexes (holoenzyme with substrate or inhibitor).''S j h e X-ray structure of the apoenzyme (EE-isozyme) was determined to 2.4 A resolution in 1976.33 The structures of the ternary complexes with NADH and dimethyl sulfoxide,^^ NAD+ and p y r a z o l e , 3 5 NAD+ and p-bromobenzyl alcohol,36 tetrahydro-NAD and dimethylamlnocinnamaldehyde37 and NAD+ and 2,4-(4-pyrazolyl)butylisoythiourea,38 have been determined to 2.9 A resolution. HLADH (EE) has been found to be a dimer comprised of two identical subunits of Mr 40,000 (Figure 5). Each subunit contains both a coenzyme and a substrate binding
485 domain. The single polypeptide chain of one subunit contains 374 amino acids of known sequence.^d These two subunits are separated by a cleft, containing a deep pocket. Both subunits of the dimer bind coenzyme and substrate in essentially the same manner; thus these two active sites are assumed to be the same.^s The two subunits of the dimer are linked by interactions within the coenzyme binding domains which form a core of 143 residues in the middle of the molecule. The catalytic domains are at opposite ends of the dimer and comprise 231 amino acid residues.33
Figure 5. Schematic of dimeric HLADH.^^ The coenzyme binding domain has a structure very similar to corresponding domains in other dehydrogenases and some kinases. The coenzyme binds in the cleft across the edge of the coenzyme binding domain via several hydrogen bonds, charged and hydrophobic interactions.'^^ The nicotinamide moiety of coenzyme is positioned in the active site with the A-side of the ring facing the zinc atom.^^ Coenzyme binding induces a conformational change from an open to a closed form. The enzyme may exist in both conformations, but the equilibrium favours the open conformation in the absence of coenzyme binding.i^
486 The major conformational difference between the open and closed forms is a rigid body rotation of each catalytic domain by about 10° with respect to the coenzyme binding domain. Thus, a cleft between the catalytic and coenzyme domains is closed, making the active site less accessible to solution and more hydrophobic. During this change some amino acid side chains, especially that of, Val-294 change their orientation. The side chains of Leu-57 and Val-294 which are about 15 A away from each other in the apoenzyme form, are only 4-5 A apart in the ternary complex. No new residues are brought into the substrate binding domain. The conformational change also brings the catalytic zinc 1 A closer to the nicotinamide binding domain and moves the substrate and nicotinamide binding domains closer. The interactions between the protein and the nicotinamide ring of coenzyme are assumed to be involved In Inducing the conformational change of the enzyme during coenzyme binding.18 COENZYME
174
CYS
Figure 6.
48
Schematic of ethanol in substrate binding domain of HLADH.
The substrate binding domain has two zinc atoms, one of which, the catalytic zinc, is located at the bottom of the hydrophobic substrate binding domain, 20 A from the surface of the molecule. Cys-46, Cys-174 and His-67 are ligated to this zinc which binds the substrate in a position relative to the coenzyme that facilitates direct hydride
487 transfer.""^ The function of the second zinc atom and the protein lobe that surrounds it, Is unknown. A deep pocket accommodates the substrate and the nicotinamide moiety of the coenzyme. It is 5-10 A wide and --20 A long, extending from the protein surface to the catalytic zinc atom. The part of this pocket associated with substrate binding in HLADH Is much larger and more hydrophobic than in other dehydrogenases. The substrate binding domain consists of a large hydrophobic barrel lined with non-polar side chains of Leu-57. Phe-93, Phe-110. Leu-116, Phe-140. Leu-141, Pro-295, Pro-296, lle-318 and Val-294, which are in the same subunlt as the ligands to the catalytic zinc that resides at the bottom of the barrel.33 The only polar groups in catalytic site are Ser-48 and Thr-178 (Figure 6). In the open apoenzyme form several firmly bound water molecules are located in the inner part of the pocket outside the co-ordination sphere of zinc. In the closed form of the ternary complex the coenzyme and the substrate occupy this space.''S According to the X-ray crystallographlcally determined structures of ternary complexes of HLADH the side chains of amino acid residues bordering the substrate binding domain can adopt chain conformations compatible with the volume occupied by the bound llgand. In the binding of p-bromobenzyl alcohol and the inhibitor, dimethyl sulfoxide, there are changes in the positions of the side chains of Leu-116 and Ser48.36 The conformation of Leu-116 is also different in the NAD+-4-lodopyrazole and (dlmethylamlno)cinnamaldehyde complexes.37 in the NAD+ and 2,4-(4pyrazolyl)butylisothlourea complex side chains of Met-306, Leu-57, Leu-116 and lle318 assume different conformations than in the HLADH-NADH-dlmethyl sulfoxide complex.38 6.5.
Previous Models for the Substrate Binding Domain of HLADH
Several models have been developed for the substrate binding domain of HLADH from kinetic studies and/or X-ray crystallographic data. The substrate binding domain has been analyzed using diamond lattice and cubic section models (Figure 7). The first attempt to describe the topology of the substrate binding domain was by Prelog In 1964 In terms of the diamond lattice model.^"' This work was refined by Jones et al. In 1976 and 19778.42,43 and Horjales and Branden in 1985.^4 The first cubic section model of HLADH was developed by Jones and Jacovac in 1982.'^5 other types of
488 models have been developed by Ringold et al. in iges,'*^ Nakazaki et al. in 1980,^-^ Duller and Branden in 1981^8 and Lemiere et al. in 1982 (Table 2)."^^ The diamond lattice model (Figure 7) was developed using six-membered ring ketone substrates. The determination of forbidden and undesirable positions was achieved by analysis of the relative rates of reduction of a series of cyclohexanones and decalones of known absolute configuration.8 The geometry indicated at the C-0 centre was considered to resemble the structure of the alcohol rather than that of the ketone in the transition state. It was assumed that all substrate molecules bound with oxygen in the
("-
\\A] O — undesirable position # - forbidden position
0
OH - forbidden cube
Figure 7. Diamond lattice and Jones cubic section model. same orientation and direction of the forming C-H bond. The back of the lattice section was thought to be a flat coenzyme binding site and the lower and left sides of the lattice were thought to be bounded by the enzyme. The oxidation and reduction was assumed to be forbidden if binding of a substrate superimposed a group in one of the enzyme occupied locations. If the group occupied an undesirable location, the rate of reaction would be very slow. Horjales and Branden (1985) constructed a diamond lattice model by docking cyclohexanol and its monoethyl derivatives into the experimentally determined active site of the enzyme (X-ray crystallographic structure, 1982), using computer graphics and energy minimization methods."*"^ The lattice positions were classified as allowed, forbidden or boundary depending their distances to protein atoms (Figure 8). The
489 lattice positions were considered as allowed (A), if all distances to neighbouring atoms are larger than 2.9 A, forbidden (F), If any distance to a fixed atom Is less than 2.6 A or boundary (B). Boundary regions were sub-grouped into B 1 , consisting of those positions that Interact only with side chains of Leu-57, Leu-116 or Met-306 and B2 comprising all other boundary positions. The volume available to substrates was similar to the substrate binding domain derived from analyses of previous kinetic data of the methyl cyclohexanones (Dutler and Branden, 1981^^) and the diamond lattice model (Figure 9a).s Using this method, Horjales and Branden extended the lattice model to extend the limits of definition of the substrate binding domain (Figure 9b). Table 2.
A summary of previous models and methods HLADH, presented
chronologically Author
Year
Models
1. Prelog
1964
2. Ringolde/a/.
1965
3. Jones et al.
1976
4. NakazakI ef a/.
1980
5. Dutler & Brandon
1981
6. Jones & Jacovac
1982
7. Lemlereet al.
1982
the diamond lattice model, using kinetic data composite structures of satisfactory and unsatisfactory substrates In front and side views the refined diamond lattice model the C2 -ketone rule oxidation and reduction cyclohexanol rings superimposed and compared; kinetic data; X-ray of apoenzyme the cubic section model (manual) "flat" cyclohexanone model
Substrates cyclohexanone and decaione derivatives 3- and 4-alkyl cyclohexanones, 10methyl-2-decalones cyclohexanone and decaione derivatives the cage-shaped C2ketones 2-,3-,4-alkylcyclohexanol with alkyl groups, methyl, ethyl, l-propyl and t-butyl. alkyl cyclohexanones 2-,3-,4-alkyl cyclohexanones
8. Horjales & Branden 1985
the diamond lattice
cyclohexanol and Its
model, using docking
monomethyl derivatives
& computer graphics
490
asfi(\
utnoi
Figure 8. Stereo diagram of the substrate binding domain using the substrate docking method.
Figure 9. Figure 9a: A comparison between two diamond lattice models. Positions A-J are derived by kinetic studies as forbidden or hindered. Positions K-R represent boundary or forbidden positions defined by Horjales and Brandon. Figure 9b shows the extended diamond lattice model. In 1982 Jones and Jacovac mechanically constructed a cubic section model using Framework Molecular Models.-* The cubic section model was conceived because
491 substrates other than those containing a cyclohexyl ring, e.g. cyclobutanones, and cyciopentanones were not easily fitted to the diamond lattice model. The lattice was based on sp^^-hybridised carbon lengths and angles. Thus, accurate predictions for numerous substrates were difficult to make. Models that resemble the cubic section approach have been developed for mono-oxygenases (Johnson in 197850) and microbial reductases (Nagazaki etal. in 198051). Jones and Jacovac employed the kinetics of alkyl cyclohexanone reductions (Duller et al. 1977,1981 and Lemi^re et al. 1980) for the estimation of forbidden and limited access volumes. In addition, they used the X-ray derived structure of the apoenzyme, the enzyme-NAD complex and several ternary complexes to derive the shape of the substrate binding domain (Figure 10a). The model, used 1.3 A3 cubes and the C-0 function is defined as an alcohol as in the diamond lattice model. The oxygen of substrate Is assumed to ligate to the catalytic zinc and hydride delivery or abstraction occurs from the front. Jones and Jacovac defined forbidden and limited access regions as A1-2, El-3, G4-5, L4-6, M7-8 and Q7-9, the left-hand halves of B1, H4 and J4, P7 and the right-hand halves of B1, H4 and N7 as well as cubes 0 9 , P8, P9. Binding in the limited access area results in a reduced rate of reduction of substrate. Cubes below the plane of the paper reflect the character of cubes immediately above them. The allowed region was suggested to correspond to the substrate binding domain of HLADH as identified by X-ray (Figure 10b). However, the boundaries between the allowed, limited and forbidden regions were assumed to be flexible due to the movement of amino acid side chains during substrate binding. The forbidden cube
y/ipg/^ y^ y%
**.^T"" "^y^
[M
N
o
p
Q
R
G
H
1
J
K
L
A
B
0
D
E
F,-- ..t.
5
>
-aC
^^
U
Figure 10.
U(F1)
£9
8
/ 4
2
»
3J
V
Leu57 Ser 48
\Z^ ^
OH
Figure 10a, Jones cubic section model. Figure 10b, Correlation with X-ray crystallographic studies.
492 E1 corresponds to the Phe-93 location; K4 and Q7 to lle-116; E2, E3 and K5 to Phe110; 0 9 , P9 and Q9, to lle-318; B1 to Ser-48; G4, M7 and half of the cubes, H4 and N7 to Leu-57 while J4 and P7 are limited because of the nicotinamide coenzyme. O
6 (2R)-]
,"^2- tt::p=^ — H - ^ : i i j 2 : i £ ! i ^ o I N7
07
P7
Q7
H4
14
J4
K4
B^
El
B1 >^1
KJ-^H
O
II
HD' ^ H
6' (2S)-1
HLADH (2S,2S)-2
I
iV^^X
Figure 11. Prediction of stereochemistry of reduction for 2-alkyl cyclohexanones by Jones cubic model. The direction of hydride delivery from NADH is indicated by the arrows > and > for reactive and unreactive product or orientations, respectively. Only conformation IV can bind without penetrating forbidden regions.
493 The prediction of product fit into the HLADH active site was conducted separately for each enantiomeric product, using the mechanical model. For the product to fit well, the bound substrate must not occupy forbidden positions. Penetration of substrate Into two or more cubes of limited access was judged to be equivalent to a forbidden interaction. The Jones group have applied this model to more than 50 different cyclic and bicyclic compounds (e.g. Figure 11). The predictions for both reductions and oxidations use the same model and are reliable. 6.6. Present Cubic Section IVIodei for HLADH 6.6.1.
Generai
The goal of the present study was to develop a computer-based cubic section model of the substrate binding domain of HLADH. It was considered that the Jones cubic section model could be refined by use of computer assisted substrate overlay in combination with kinetic data on a wide variety of substrates. As in the Jones approach we used the alcohol products as the surrogate substrate structures. Thus, we determined the low energy conformation of alcohols produced from ketones that have been reported to be reduced by HLADH and for which comparative kinetic data vs cyclohexanol could be calculated. As well, we determined the preferred conformations of all alcohols that would have been produced from ketones subjected to but falling to undergo HLADH reduction. These calculations utilised molecular mechanics (MACROMODEL) and yielded accurate co-ordinates for all atoms in each alcohol. Where enantiomeric or stereoisomeric alcohols were produced or capable of production, the co-ordinates of each were calculated. Alcohol products were assigned a priority number based on their rate of production vs cyclohexanol. Relative reduction rates, enantiomeric excesses of alcohol products, extent of conversion of substrates, yields and absolute configurations of alcohols were used In calculating the priority number for each substrate with the faster reacting substrates receiving the higher priority numbers. Co-ordinates of atoms in the energy minimised alcohols were transferred to ENZYME, a program that allowed the structures to be identically oriented in a cubic section model. Using ENZYME program the energy minimised substrates were placed in a Cartesian co-ordinate (x, y, z) system with C-0 aligned with the carbon at the origin, the oxygen on the negative y axis and the ahydrogen the yz-plane.
Conducting this operation on all alcohols gives a map of
acceptable and forbidden cubes according to the average priority numbers for each
494 cube. Forbidden areas were assumed to be occupied or blocked by amino acid residues or coenzyme. The model is directly comparable to that of the Jones group in that we have used cubes of 1.3 A on a side and placed the carbon-oxygen bond as well as the carbon-hydrogen bond of the carblnyl carbon In the same orientation. We have compared the present model with that of Jones as well as with the X- ray crystallographic structure of the enzyme. 6.6.2.
MACROMODEL and ENZYME Programs
MACROMODEL and ENZYME allow facile structure entry, energy minimization, reorientation, atom to cube assignment and calculation of average cube priority values for each cube.
MACROMODEL, developed by Clark Still at Columbia University
employs a recent version of Allinger's popular force field minimization routine. Each molecule is described in the program according to the atom types It contains and the connections between atoms. The energy of each conformation of each structure is calculated by evaluation of the Van der Waals, bond stretching, bond angle bending, torsional and dipole interactions. In Appendix 1 there is a description of those parts of MACROMODEL that were useful in this work. ENZYME SITE PREDICTION SYSTEM V 1.6
• • Bi m •
-
Forbidden Priority < 0.5 Priority < 1 Priority < 5 Priority < 10
•
- Priority >= 10 Layer 1
Cube file is col 3.cf
READ CF READ DATA ADD TO CF REMOVE CF ALIGNMENT ROTATE ON X ROTATE ON Y ROTATE ON Z PREDICT DISPLAY CF ROTATE BONDS
Press
PRINT CF SAVE EXIT CSl.30 CORNER DISCOL 6
Figure 12.
Menu of the ENZYME program. One layer of constructed substrate
binding domain is presented.
495 The ENZYME program was developed at Simon Eraser University In Pascal on a VAX 750 using an IBM personal computer with an enhanced graphics adapter and graphics tablet running a VT 100/Tektronix 4107 terminal emulator. The source code program is available from the authors. It is fully compatible with the MACROMODEL program and was designed to be easily transferred between computers. ENZYME allows construction of cubic section substrate binding domains for enzymes other than HLADH. The program executes various procedures through menu driven processes. Representative procedures are shown in Figure 12 are read the product file, add the information for the molecule to the cube file, remove information for the molecule from the cube file, align the molecule in space, rotate about an axis, predict the averaged priority for the product, display cube file data, rotate about a bond, change the size of the cube, shift the origin from the center of the cube to the corner, print the cube file data, save the cube file and exit the program. An additional program, COMPARE, allows plots and cube by cube comparison of two constructions of substrate binding domains. COMPARE was also written in Pascal on a VAX 750 using an IBM personal computer configured as above and the source code is available from the authors. The documentation of ENZYME and COMPARE menus are presented in Appendix 2. The details of the use of the programs, MACROMODEL and ENZYME are described in Chapter 7.5. 6.6.3. Criteria for Choice of Substrate Surrogates As in the Jones protocol the cubic section model of the substrate binding domain of HLADH were constructed using structures of alcohol products rather than ketone substrates. The alcohol products were originally chosen by the Jones group because the transition state the geometry for the reduction was considered to resemble that of the alcohol rather than that of the ketone. The relative rate of reduction of substrate vs cyclohexanone for each ketone was required to be known. Furthermore, configurations of alcohol products, enantiomeric excess values, yields and % conversion of substrate required for calculation of the priority number for each enantiomer of product should be measured under comparable conditions (i.e. pH, temperature, concentration of enzyme, coenzyme and substrate, etc.). According to Alderweireldt et al. (1988) HLADH models are valid only for reaction conditions used in the reactions from which the models are constructed.52 Furthermore, the model is only reliable if the reactions have been conducted under kinetic control.
496 Table 3. The substrates used in construction of the active site picture. OH
OH
OH ,
OH ,
^48.53
2^.53
38
OH
OH
OH
g26
754
g54
OH
OH
^353,55
^^53,55
OH
OH
^48
5S4
g54
OH
OH
OH
,0^
„26
^^26
OH
cis & trans
cis & trans
^^
trans
2 1 ^ ' 22^=*
23^'
cis & trans
,ge
-.8 ,78
cis & trans
,«8 ^g8
^g„
2o„
OH trans 24"
2 ^
2 ^
05 OCT A„ ^ 27®
32
28®
29^
33
34
X"
30^^
31®
35
36 OH
OH HO
^
HO 37
38
39
57
40
41 OH
^ 4 OH
OH
42"
43
,58.56
OH
44
45^
497 Using these criteria 45 substrates for which experimental information was available for reaction at pH 7 were chosen to construct the model (Table 3). There is a significant amount of literature data for substrates of HLADH which lack information on enantiomeric excess values and absolute configurations of products and where the relative rates of reaction have been measured under different reaction conditions (e.g. pH 8.5). Substrates falling Into this category (46-69, Table 4) were not used. Heterocyclic bicycllc substrates 70-73 were only used for testing.
O
Example 1: (±)
OH OH
O
Example 2:
OH
OH
ir &
^ (±)
OH
OH OH
X::^oH^^^
\::^
^=^/X:::poH
Figure 13. The possible conformations of two substrate surrogates.
498 Table 4. Product structures used in testing model for prediction of stereoselectivity. OH
OH
OH
OH
OH
46^^ 46''
4/^ 47'
48^
49^°
50^°
OH
OH
OH
OH
OH
6 ^ 6 ^ O^ Cx. 6^ .60
60
52'
51^ OH
53
OH
6
OH
.60.49
g^60.49
62
OH
^
-OH
^60.49
^H
6< 6: A^.o^ 67^
66"
HO"^-^^
71
68"
HO-^^T"^
72'
69"
70"
HO-^^-^TT"^
73'
Structurally rigid substrate surrogates were added to the cubic section model before more flexible molecules as the following order signifies: pentacyclic, tetracyclic, tricyclic, bicyclic ketones, trans/cis-decalones, methyl cyclohexanones and alkyl cyclohexanones. The hydroxyls of cyclohexanols were oriented axlally with respect to cyclohexyl rings consistent with the Jones protocol and with the observation that
499 reduction of conformationaily locked trans-decalins (16,18, 20, 22 and 23) produced exclusively axial alcohols. All low energy conformations of conformationaily flexible alcohol products were added included (Figure 13). 6.6.4.
Calculation of Priority for Alcohol Products
The priority number for an alcohol product was determined by the relative rate and the stereoselectivity of reduction. The priority number for each substrate surrogate was calculated from the rates of production of each enantiomeric alcohol product using the total relative rate of reduction vs cyclohexanol and the enantiomeric ratio (E) formula derived by Sih (Table 5).^^ Table 5. Formulas for calculations of the priority numbers for product enantiomers.
ln[1-c(1-fee(P))] *=~ln[1-c(1-ee(P))]
Sih's formula For product 1:
(1) (2) (3)
For product 2 :
P+ = R ( Y , + Y 2 ) ^ E 2 + I )
p-=f^(v^)(E?n-) where
(4)
(5)
E = the enantlometric ratio, the discrimination between two competing enantiomers by enzyme c = the extent of conversion of racemic substrate e e ( P ) = the optical purity, the enantiomeric excess of the product P = priority number for major enantiomer p = priority number for minor enantiomer Y = yields of products R = relative rate vs. cyclohexanone
The relative rate for cyclohexanone reduction was set at 100. This would give, for example, a total relative rate of 24 for the reduction of 2,3,5,6-tetrahydro-2-
500 isopropylpyran-4-one (precursor of 7, Figure 14)7 ENZYME contains a subroutine to calculate the priority numbers. It will request ail necessary values for the equations (Table 5). The ratio of enantiomers (major or minor) are to be given by the user. The priority values are calculated and displayed by using the PREDICT opWon of the menu. The priority number, 22.6 is given for the major product, (2S,4S-7). For the enantlomer, the priority number is near zero because the enantiomeric excess for this reaction is 1.00 (Figure 14). OH
QH
0.r
R = 24
c = 0.5 (2S,4S) Yi=32 ee=1.00
(2R,4S) Yi=2 ee=0.28
(2S.4S) Product 1:
P+ = 22.6 E = 15200
'
(2R.4R)
p. = C
(2R.4S) Product 2:
E = 2.3
I
P+=1.0
(4)
p. = 0.4
(5)
>
(2S.4R)
Figure 14.
(2)
^
Priority number calculation for enantiomers of 2,3,5,6-tetrahydro-2-
isopropyl-pyran-4-ol.^ 6.6.5.
Construction of Substrate Binding Domain for HLADIH
A global energy minimised conformation using MACROMODEL was obtained for each compound to be used In construction of the model. The torsional angle constraint was set at zero degrees for the atoms needed in this orientation then the minimization process was executed using the Block Diagonal Newton Rapson minimization method, BDNR.
Compounds to be included in the model were reoriented according to the
Jones protocol, using the MACROMODEL ANALYZE mode (ALIGN X, ROTATE Y options). The C-0 bond is aligned with the carbon at the origin, the oxygen on the negative y axis and the a-hydrogen in the yz -plane (Figure 15). The coenzyme is assumed to be in front of the origin and the catalytic active zinc atom is in the lower portion, directly ligated to the hydroxyl group of the substrate.
501 Each compound data file was transferred in ASCII format from MACROMODEL to the ENZYME program by execution of MM FORMAT on the file produced by MACROMODEL. Compound files in ENZYME are called for entry Into cube files by using READ DATA option. At this point the program requests the priority number of the compound which was calculated by the process, explained in Section 7.4. Using the ALIGNMENT option each compound was aligned with reference to the axes with the carbon on the origin (0, 0, 0). It was helpful at this juncture to check the orientation of each structure by rotation about the y-axis by ±10 degrees. Correctly minimised and oriented structures were added to the cube file of a chosen name using the ADD CUBEFILE option. After each stmcture was added the cube file was saved. The model produced by this process could be viewed, using the DISPLAY CUBEFILE option. To obtain the co-ordinates of the cubes and average priority value of each cube the PRINT CUBEFILE option was used. Addition of a structure to an empty cube file assigns each occupied each cube of the model the calculated priority of the structure. If atoms of a second structure occupy some previously marked cubes the average of the priorities for the entered structures will be calculated for these cubes. When a structure known not to be produced (i.e. Its priority is zero) is added to the model all cubes not previously identified as being occupied and which are occupied by that molecule will be marked forbidden. The forbidden classification will be removed If a subsequently added structure has atoms occupying those cubes. The program does not use priority values of zero to calculate average priorities. Addition of many structures to a cube file creates a cubic section model in terms of allowed (with averaged priority numbers) and forbidden (priority 0) cubes. Each cube is identified according to the Cartesian co-ordinates (x, y, z) of its corner farthest from the origin. The modelled volume contains several layers of cubes of user defined size. ENZYME allows average priority values of cubes to be viewed in different colors (represented as shades in Figure 12). Display of cube priorities in two colors can be chosen. Green is the default color for allowed cubes and red for forbidden cubes. A hard copy of the average priority values for each cube can be obtained by using the PRINT CUBEFILE -menu option. The method allows openended improvements since new structures can be added to refine cavity topology.
502
^
Reaction
_^h^£L,
J^
(±) rel rate vs cyclohexanone = 26 conversion of the reaction * 0.46 an enantiomeric excess of product = 0.64 [^ a yield of product = 39
E - 7.76
Computer simulation H
(0-^
MM2
A T H -^--'- y^H
DRAWN
I
PRIORITY = Acceptance ranking
REORIENT
OH 2.97
23.03 (01lY
I
yo
z:
m
I cube - atom match P = cube acceptance ranking
cube acceptance ranking = P
A = average acceptance ranking
I repeat for additional substrate Figure 15. Procedure of defining topology of the substrate binding domain of HLADH.
503 Using the same equivalently oriented structures 12 different cube files were constructed (Table 6). The effects of location of the origin in the corner or In center of the center cube, size of cubes (1.3 A or 0.65 A) and inclusion of hydrogens in the structures were studied. Special cube files, coded co13*, co065*, ce13* and ce065* were constructed without the hydrogen on oxygen (Table 6). Usually this hydrogen can occupy two side by side cubes in layer -1. This is due to very slight differences in orientation produced during the minimization process. Table 6. Twelve different cube files constructed. orii jjln corner center
Icode of cube file
1. co13
X
2. CO065
X
3. 0013*
X
4. CO065
X
5. co13h
X
6. co065h
X
X
X
X
X X
X
X
X
11. ce13h
X
12. ce065Hi
X
X X
X
X
10. ce065*
6.6.6.
X X
8. ce065
1
X
X
X
X
a-H&H on SL b s t r a t e s oxygen g-H aii hydrogens
X X
7. ce13
9. e e l 3*
cub ) size 1.3A 1 0.65A
X X
X
X X
X X
X
Model for Substrate Binding Domain of HLADH
A goal of this work was to refine the cubic section model developed by Jones"^^ using computer modelling. Thus, equivalent orientations of compounds, cube size, 1.3A. and origin location were used to construct the cubic section models of HLADH. Because the jacks and plastic tubing of the mechanical model occupy space, Jones changed
504 the edges of cubes C, D, I, and J in layer 0 to 1.4 A. Computer modelling allowed the Present Model (co13) Jones Model
layer 4
layer 3
layer 2
M^ N
0
P
Q
R 1
p
H
1
J
K
L
r
B
c p
E
F
u
u
u
u
u
u
1
505
layer 1
[N
0
H
1
B
c p
u
U
^M J
u
layer 0
0.02
^ ^
1
layer-1
4
34
1
lofH
I1
45 10 9 11
506
layer-2
fA
N
0
P
Q
R1
r
H
1
J
K
L
r
B
c p
E
F
u
u
u
u Cubes:
u
u
n
•
|58j allowed in present model I
>
(5 = averaged priority number, 8 = number of atoms)
I allowed in Jones model forbidden
1 1
cube size 1.4 A
forbidden or limited in Jones model limited in Jones model allowed for some 3-alkylcyclohexanols or heterocyclic bicyclic alcohols (usually forbidden) allowed for some 4-alkylcyclohexanols (before unknown) Figure 16. Cubic section model for the substrate binding domain of HLADH: Jones model (left) and the present (co13, right). Jones assigned each cube alphabetically and we have used his convention to allow comparison. The priority numbers for each cube were rounded in the present model. size of ail cubes to be of the same. The construction of the original cubic section model used only the kinetic data of alkyl cyclohexanones. In the present model 166 different conformations of alcohol products and potential products were used to construct each cube file. Conformations of 55 had positive priority values, while 111 represented non reactive conformations.
In both the Jones and the present models substrates
contained only one carbon bound hydrogen which is the a-hydrogen and a hydrogen on oxygen, in our model other hydrogens of modelled structures were deleted after minimization. The previous division of cubic space into allowed, limited and forbidden layers spaces has been refined in that ENZYME generates individual acceptability rankings for each cube (Figure 16). ENZYME gives a printed list of the cube file: the contents of each cube, the average priority of cubes and the total number of atoms eventually in each cube. For model c o l 3 the origin is in cube OD (cube D in layer 0),
507 the oxygen In cube OD-2 (2 cubes under cube D in layer 0 ), the a-hydrogen In cube -1 D (cube D In layer -1 ) and the hydrogen on oxygen In cubes -1C-2 or -1D-2. In both the Jones and col 3 models the allowed area opens most significantly to the left of the origin. This can be seen more clearly by the present model because layers 3
Table 7. Comparison between cubes in Jones and col 3 models. 1 layer, 1 the code Jones model Present model the contents of cube of cube col 3 & priority ( ) layer -1 H K 0
F F F
34 4 0.02
substrate 30a(39) 30b(4) 31(0.02)
layer 0 G M N H e J Q K
o aM-aR 1
F F F&L F&L L L F F A ?
0.2 0.8 0.01 1 2 12 1 1 0.2 17 1 For A*
! 1
15a(0.2) 2b(0.57).14a(0.98) 45b(0.01) 10 substrates 33d, 41b, 90a. 30b 15 substrates 45a(1.29) 38d(0.27),37d(0.05) 38c, 30a *aO: 70a
layer 1 A & uA G M H N uB o
J p Q K E uE F—2 aN-aQ
F F F A A AorL A A. F or L F F F F F ?
For A* 0-2 unk 2 2 2 0.5 2 5 4~ 0.1 13 1 0.4 For A*
*52a, 53b, 55a, 48a I5a(0.2) 38a, 37a, 40a 37c. 41b, 44b 12a(3.6),39b(1.01) 37c, 37d, 42b 11 substrates 37c, 37d, 42a 44a(94.9),37d(0.27) 38b(0.08),37b(0.1) sulphur-compounds 39a(1.49) 7d(0.43) VO to 73
1
508
layer 2 K
F
Q E
F F
uE
F
uA bG uC B-2 N G 0 P
A A AorL A A A ForL ForL
0.4 unk 2 0.2 2 2 1 F A* 13 4 For A*
45a(1.49),45b(0.57) 38d(0.87),37d(0.05) 38b(0.08). 44b(5.13) 45b(O.Oi) 7d(0.43), 6d(0.08), 5d(0.26) 2x120(2.39) 2x120(2.39) 39b(1.01) *58 110(23.2). 120(2.39) 17a(7.5). 41b. 19a 60a, 63b. 66b, 65a
layer 3 bG bA A B
? ? ? ?
61 61 31 11
C D C-2 D-2 D-l C-l J 1 aP aQ E
? ? ? ? ? ? ? ? ? ? ?
3 4 28 28 A* A*
1
1
A* A* A* A* For A*
8a(120.5), 120(2.39) 8a(120.5). 120(2.39) IOo(39.3). 110(23.2) 18a(3.0), 6a(14.8), 7a(22.6). 20a(3.5) 18a(3.0), 20a(3.5) I9a(4.0) 80(27.53) 80(27.53) *58a *59a 58a, 58b. 59a, 59b 58a, 59a 66b, 65a, 63b 65a 70-73
j
layer 4
1
4c
?
A*
61a. 63a. 65b. 66a
and 4 are more fully defined. Due to the greater number of compounds used the allowed area in the present model is slightly larger than in the earlier version. A list of average priority values for eaoh oube are given in Appendix 3. comparison of each cube in the two models is presented in Table 7.
The detailed
509 The cubes with priority numbers of 2 or lower are considered boundary areas (Table 2). They are allowed for some substrates and forbidden for others. The priority of structures that occupy these cubes is usually very low. Some bulky structures were found to occupy low priority cubes G, N, Q and K in layer 0, cubes G, K and uE in layer 1 and cubes K and E in layer 2. These are forbidden in the Jones model. Bulky substrates are assumed to change the orientation of amino acid side chains lining the substrate binding cavity. It is suggested that except for bulky structures these cubes will be forbidden as suggested by Jones. The high priority value of cube 1E (13.0) is caused mainly by sulphur containing substrates. In every case C2 of these heterocycles affects this cube making it allowed. The high value of cube 1Q (48.0) is due to one cage-shaped substrate (44a) with a priority value of 94.9. Another substrate (37d) occupying the cube has a priority value of only 0.27. It is suggested that this cube also belongs in a boundary area. Cube, OM, forbidden in the earlier model, is a boundary area with average priority value, 0.8 in the present (col3) model. The cube contains two atoms of 2-ethyl cyclohexanol (2b) and its sulphur analog (14a). Cubes 1H, 1N, 10 and U are allowed in the earlier model and each have an average priority value of 2 or lower 0013. They are occupied by atoms of bulky substrates. In the earlier model cube OJ was limited access, while In the present model its average priority value is high (12.0) and it contains 15 atoms of as many substrates. Cube IP, with a priority value of 5 (occupied mostly by bulky substrates), is a boundary area In both models. Cube OO, with a priority value of 17 contains bulky substrates (38c, 30a). Usually areas above cubes OM to OR are forbidden in col3. This volume was not defined In the Jones model. According to the Jones model regions below the origin reflect the character of cubes immediately above them. Thus, those cubes below limited or allowed cubes were assumed to be limited access. Cubes, 2D-2, 2D-3, 2E-2, 2E-3 and 2E-4 should then be limited although they are forbidden in col 3. The average priority value for cube 1M is undefined with the data we used. It could belong to a boundary area as does the neighbouring cube IN. Cube 2B-2 could be allowed if more data were available. Cubes 1F-2 and 2uE have low priority values and are suggested to be in a boundary area. Bridged substrates, 30a, 30b and 31 effect some cubes in layer - 1 , making them allowed. It is possible that these substrates change the orientation of amino acid side chains or coenzyme near this layer to allow binding. The cubes 3bA, 3bG, 3C"2, 3C-3,
510 2bA. 2A-1, 2C-2, 2C-3, 2B-3, 1B-2, 1A-2 and 1A-3 are occupied by atoms of phenyl groups of one or two substrates (8,12, Figure 16). Some structures were examined which were not included in the cubic section model used for testing. For example, linear and branched 3-alkyl cyclohexanols can change some forbidden cubes in col 3 to allowed. Also 4-alkyl cyclohexanols were found to extend the allowed area In layers 2, 3, and 4. These substrates were not used in the construction because they were studied under different reaction conditions or no values of enantiomeric excess were available.
Addition of substrates containing
hetereocyclic bicyclic rings (70-73) would change cubes OaO, 1aN, 1aP, 1aQ, and 3E to allowed (Figure 16). After construction of model col 3 wherein the origin was on the corner of a cube It was of interest to visualise the effect of placing the origin on the center of a cube (eel3, Figure 17).
This process was executed for the same set of structures used for
construction of the corner centred cubic sector model. Placing the origin at the center of a cube alters border areas and changes the average priority values on most cubes as well as the number of substrate atoms in each cube. Appendix 4 gives the printed list of cube priorities for the center cube origin model (ce13). The effect of cube size was also studied. Thus, the cubic section model was constructed with the same set of substrates as model co13 assigning the origin to the corner of the center cube but with a cube size of 0.65 A. The printed list of the cube priorities for this model (co065) is in Appendix 5. Altogether 14 different layers were defined. A combined view of both co13 (1.3 A) and co065 (0.65 A) models allows refinement of the 1.3A model (Figure 18). For example, only half of the cubes OK, OH, IK, I E , 2E and 2K allowed in the 1.3A model are only allowed in the 0.65 A model. The cubes OH, ON, I N , and 1H are regarded to be on a boundary area. Jones assumed that the right half at the cubes OB, OH, and ON were limited and half were forbidden. The present analysis allows clearer definition of the border of forbidden and boundary areas.
511
3.3^
o 28'
layer 5
p
61^
layer 4
23«
6^
3
61^
r
21
6l2
18«
l'
4^
5*
0.01
23^
0.22|
10^
"^
4'^
15 1 11^= 3='
iiH 9«
o
71 12H 4^
12 1
^^0.24^
2
4^
16
4'
16
2
28'
layer Z
layer 3
^ 4 11 ^0.5 11 0.5= 52 ^
1
11
12 5 8 4 0
15 6
p ^
3 P
11 J2
1 4
layer 1
layer 0
512
59
O
iQ> .55 10
layer-2 layer-! I
I allowed
^ ^
forbidden
o=origin
Figure 17. Cubic section model (ce13) of the substrate binding donnain of HLADH.
0
layer 4
layer 3
513
•ii
layer 2
layer 1
layer -1 layer 0
allowed forbidden
D
forbidden position in 0.65 A map
unknown
layer -2 Figure 18. Cubic section models of the substrate binding domain of HLADH using 1.3A (col3) and 0.65A (co065) cubes.
514 6.6.7.
Testing of the Models
All twelve cubic section models listed Table 6 were tested to determine which constituted the best model for prediction of substrate reactions with HLADH. The effects of the origin placement, cube size and structure of substrates were tested. In some analyses compounds that had been previously used in construction of the models were used. If this were the case the entries relating to these products were individually removed from the model before a prediction was executed. For new products the minimization, orientation and alignment processes were conducted before execution of a prediction. The PREDICT option (mouse driven) gives the average priority of cubes occupied by each atom of the test molecule and identifies those atoms in cubes of undefined and zero priority. PREDICT (pressing the second button of the mouse) will write the above information to a file with the same file name as the molecule file but with the extension '.pre'. Another way to visualise the results is to use the cubefile graphic display. The positions of each atom of the test molecule are seen as white circles in the individual layers of the model. The results of prediction process were analyzed using both the data in the prediction table (average priority value of each cube, average priority value of cubes over molecule, forbidden sites and unknown sites) and the graphic display. Generally, if the average priority value is quite high (approximately 10 and over) and no forbidden cubes or no occupied cubes with low (A<1 ) priority are present the test molecule will be formed rapidly by HLADH. If there are two atoms of test molecule In a boundary area (A< 2) formation is slow or does not occur. Figure 19 shows an example of a prediction table and a display of positions of the atoms of a test molecule in the cubic section model (co13). The test molecule is known not to be formed by HLADH reduction (the priority number is zero). Although no forbidden cubes are occupied one occupied cube has a very low priority value, 0.24. This suggests that the test molecule would not be expected to be produced by HLADH. The cubic section map for this molecule further showed that some carbons are near to forbidden cubes. It is usually better to check all visualisation methods when using ENZYME for prediction of reactivity, in practice it is found that the full prediction table gives the most accurate prediction. The cubic section model, co13 (Table 6) was used for prediction of the relative rates of formation for all 73 products in their different conformations listed in Table 3. Of these,
515
the more rigid were used in construction of the model but the less rigid (28) were not. For example. 3- and 4-alkyl cyclohexanols and heterocyclic bicycllc alcohols (Table 4) were not used in model construction. Because of the huge amount of data generated only twenty of the more interesting prediction results have been presented (Table 8). OH
layer 2 Si
IC H
9b
C I C -4-0—h-
layer 1
minimized structure
Type hoh.pre Substrate Original Priority: 0.00 Original State: forbidden ATOM ATOM # X
m - < »iiM
layer 0
H
layer -1
C 0 0 0 0 S O H C H
1 2 3 4 5 6 7 8 9 10
Y
Z
-1 1 0 0 0 0 -1 0 -2 -1 -1 -2 0 -2 0 -1 -2 1 1 -1 -2 0 0 1
CURRENT TOTAL AVERAGE #HITS PRIORITY PRIORITY 13 55 55 55 15 16 55 45 3 55
67.2 567.5 567.5 567.5 166.2 111.4 567.5 482.0 0.7 567.5
12.1 10.3 10.3 10.3 11.1 7.0 10.3 10.7 0.2 10.3
Total # atoms in molecule: 10 Average Priority of cubes over molecule: 9.3 Total # hits in cubes: 367 Average Priority for cubes aver all hits: 0.2 Number of unknown sites: 0 Number of forbidden sites: 0
Figure 19. Prediction table and part of displayed cubic section map for a test stoicture. The model col3. Only the forbidden cubes close to product atoms have been assigned.
516 Table 8. The prediction results for twent]^ test molecules using model co13. P=prlorlty A=average forbidden Test molecules Unk/Low 1 (expt) priority cube priority cube OH
.
^
,1,
10.1
OH
0
9.1
OK
0
9.1
OB
0
9.8
OE
0.05
8.5
OaM
0
7.6
OR.OaR
-2bF-'«=unk
0
7.6
0F,-2F
-2bA-i=unk
8.0 11.4
0A.-2A
0.9 OH OH
OH
OH
2.
(;V'^(46)
OH
OH
OH
3.
^
,S4, 0 52.67 OH
9.2
2E-1
0.03
10.0
2E
0.97
10.8
0 OH
\ n
517 [ T e s t molecules
P=priority (expt)
1 forbidden cube
A=average priority
Unk/Low 1 j priority cube
65.5
9.5
1A
2bA=unk
0
8.3
IF
2bF=unk
0
8.4
3E-2
2E-^
1.6
8.7
14.8
11.2
0
9.1
OH OH
^
3B"^=unk
OH
2F
2E
OH
1.2 0.02
120
10.6 1
8.6
1
2uE
2M=^unk
8.1 (28.8)
Ph
1
_''* AH
0
6.2
2F,3bF,3bL,2R
27.5
6.6(15.9)
5 cubes unk
0
6.5
5 cubes
518 A=average priority
P=priority (expt)
Test molecules
forbidden cube
, Unk/Low 1 priority cube
OH
32.6
13.9
0
9.3
12.4
10.9
0
9.3
2E-1
9.5
OH, OM
OH OH
'
OH OH
2F
y w '"' 1
1.0
1
^ OH
0
8.5
OR
0
8.9
-IB,-2A
0
8.5
-1E.2F
^ OH OH
9.
k J (62) OH
28.6 OH
1 -^J^
21.0
11.2 10.2
OK
519 [ Test molecules
1
A=average priority
P=priority (expt)
1
forbidden cube
lOnk/Low 1 priority cube
OH
J^
10.
(58) OH 1.3
7.8
0.06
8.5
3D"^
31, 3J=unk
OH
wax
3J, 2N=unk
(18)
ft
OH
3.0
10.6
0
7.7
3E
.0
1
10.1
1 laQ, laP, laO
0
1
6.7
laO, laP, laN
0
5.6
IF. OF
0
10.0
OA,OB, 1A
0
7.2
OA, OB
0
9.1
OF, OE, OL
(16)
12- k X j H
^
^ OH
1 ^^i
520 Test molecules
P=priority (expt)
00
A=average priority
forbidden cube
0
7.0
1L.0F.-1E
0
7.5
OA,-IB
0
5.2
IL.OF.OE.OB
0
5.7
OA, 08, OE
Unk/Low
1
priority cube
OH
06 06
r- A„'"' A>i,H
23.0
9.8(10.5)
3.0
9.2
U
0
9.4
U , OH
0
8.2
OP,OK
33.6
7.3(12.8)
-1H=unk
4.1
7.8(8.1)
-1 K=unk
0
9.3
U , IE-1
0
8.7
-IB-1
OH
A™ A^H MI
JO="
AV"
(30)
^
^
1
A:>OH
1
521 1 Test molecules
[
P=prJority (expt)
[
A=average [ priority
forbidden cube
rUnk/Low priority cube
r ^^''^ H O ^
1.5
8.5
1E'"'«unk
^
1.0
9.3
1B''*=unk
H
U
0
10.1
OH
0
8.9
OK, OP
13.6
9.7
IP
1.1
10.4
10
^
^ OH
17.
(42)
OH
OH
OH
L.
^
,44, OH
®
94.9
7.3(14.6)
1Q
5.1
9.9
1N.2E
522 Test molecules
A=average priority
forbidden cube
Unk/Low priority cube
(40)
19.
20.
P=priorJty (expt)
6.2
9.2
OH, U
1.3
9.6
OP, OH, 1H
9.7
IK, OK
9.4
0K.2K, U
x:b^
HO'
(70)
CO
91.3
11.5
laP, laQ
0.8
7.6
3E
81.6
7.2
OaO, laN
16.3
11.2
HOT'
xx>
Ha
For 2-methyl cyclohexanone (1), the first case in Table 8, the average priority value allows one to easily predict the main product of the reaction as the (IS, 2S) isomer, although one atom of the substrate occupies a low priority cube (OH).
The
enantiomeric (1R, 2R) product may be formed in small quantities, but formation of the other diastereoisomer (IS, 2R) and its enantiomer (1R, 2S) are clearly less likely due
523 to occupation of forbidden cubes. Our results agree with Jones prediction results for 2alkyl cyclohexanols (Figure 11). For test molecules cited In entries 2-4, 9, and 10 the assigned priorities are more difficult to compare with experiment because different reaction conditions were used in measuring their relative rates of formation. In entries 2 and 4 although many cubes of unknown priority are found, the calculated average priority values quite cleariy predict the relative proportions of products. For entry 10 cis/trans isomer ratios cannot be predicted. Only for 4-methyl cyclohexanols (entry 9) do the priority values predict cis/trans isomer ratios In agreement with experiment. The model (co13) accurately predicts preference for product formation from heterocyclic substrates with alkyl substitution. Good examples are entries 5, 7, and 8. If a phenyl group is appended to a heterocyclic ring predictions are less accurate because of the number of cubes of undefined priority occupied by this residue (entry 6). For bicycllc molecules (entries 11,12 and 13) the model accurately predicts which isomers which are not formed. Removal of bicyclo[3.2.1]octan-2-ol, entry 15, from model 0013 renders some cubes in layer-1 undefined priority, thus the priority values calculated for this case do not accurately predict exo/endo isomers. For bicyclo[2.2.1) heptan-2-ol, entry 14, priority values do accurately predict exo/endo isomer > 1 . Bicyclo[3.2.1]octan-2-one may effect the orientation of some amino acid side chains and the coenzyme which allows it to react with HLADH. The stereochemical course of reductions of some cage-shaped molecules (entries 16-19) is not well predicted. For entries 16 and 19 the products which are not formed (priority value is 0) occupy many low priority cubes. The major product with priority value 91.3 in entry 20 (new substrate) has a higher average priority value compared to other conformations but two atoms are in forbidden cubes. Based on this it can be assumed that these cubes could be allowed for some substrates instead of forbidden as shown in model co13. Models in which the origin was moved to the center of the center cube, the cube size was reduced to 0.65 A and hydrogens were added to the compounds used to construct the model and to the test compounds were next examined with 15 substrates to determine if improvements were achieved by these model modifications (Table 6, 9 and 10). Table 9 shows the predictions made by models of 1.3A and 0.65A cube size with the origin at the corner of the center cube with and without added hydrogens. Table 10 shows results obtained using the same models constructed with the origin in the center of the center cube. The average priority values of cubes over the substrates as well as the number of unknown sites and forbidden sites obtained for each substrate from each model were compared with experimentally derived priority values.
524 Table 9.
Prediction of acceptance by HLADH using cubic section models with 1.3A
or 0.65A cubes with origin at the corner of center cube, with and without added hydrogens. Average prority value, unknown and forbidden sites. Test molecule OH
P(expt)
32.6
co13
CO065
13.9,0,0 11.5,0,0
0013*^ C O 0 6 5 * co13h i 5o065h
14.4.0.0 11.7,0.0
11.4,0.0
10.5,0,0
9.2.0,1
8.0.0,0
7.9,0,3
11.0,0,0 11.1.0.0
11.1.0,0
11.2,1.0
9.2,0,0
9.9,0.0
7.5.0.0
10.6.0.0 10.3,1.0
10.2,1.0
9.3.5,0
7.5.0.1
7.2,0,3
8.0.0,2
7.2.0.7
10.1.0,3 7.3,0,4
6.9.0,8
5.5.0,14
OH OH 0
n
12.4
9.3.0,1
9.5,0,1
10.9.0,0 11.1.0.0
9.4,0.1
OH 0
9.3.0.0
9.5.0.0
9.4,0,0
OX'"' OH
J^
10.6.0.0 10.4.1.0
7.5.0.3
0
7.7.0.1
0
10.1.0.3 7.6,0.4
0
6.7.0.3
7.5.0.3
6.6.0.3
7.4,0,3
5.9,0.7 ! 4.6,0,13
33.6
7.3.1.0
6.8.3.0
7.5.1.0
6.9.3.0
5.0.0,0
3.7,10.0
4.1
7.8.1.0
7.8.3.0
7.5,1.0
7.5.1.0
6.5,0.0
3.7,12.0
0
9.3.0,0
7.2,0,4
9.1.0.0
6.8.0.4
8.2.0.0
6.0,0,6
8.8.0,0
5.6,0.4
8.8.0.0
(30)
/Kk°"
AT 1
3.0
1
^
/k^H
0
5.3,0.4
8.4.0.0
6.2,0,0
1
525 P(expt^
Test molecule
co13
CO065
co13*
CO065*
co13h |:o065h 1
^=fV^ (41) 1—1 ^
9.7
11.0.0,0 16.6,0,0
0.3
8.1,0,0
11.0,0,0 17.1,0,0
9.5,0,0
10.2,3,0
7.8,0,0
10.2,0,0
6.5,5,0
12.4,0,1 5.5,0,3
9.5,0,0
4.1,0,7
8.9,0,0
9.6,0,0
7.0,0,5
n 6.7.2,0
6.3,2,0
^ 0
12.1,0,1 5.8,0,3
^
1
*
_ X)H
1
0
1 9.0.0,0
9.2,0,2
9.1,0,3
J
In the first entry of Tables 9 and 10 predictions agree with the original model (co13), except for the enantiomer of the minor isomer (priority 0). For this compound ENZYME gives one or more forbidden sites for models coded co13h, co065h, ce065, ce065* and ce065h. In models co065h, ce065, ce065* and ce065h (Table 6) the priority values obtained do not accurately predict isomer preference. Entry 2 In Tables 9 and 10 is a good example of a case in which priority numbers accurately predict the outcome of reduction. For the bicyclic substrate (entry 3, Tables 9 & 10) the interesting result is that models with a 0.65 A cube size give many forbidden sites for endo products. In the case of cube file, eel 3 the same result was found. Many forbidden sites also are given for the enantiomer with a priority number of 0 in entry 4. We conclude that there are no significant differences between the predictive value of cubic section models wherein the origin is at the center or the corner of the center cube. Reducing the cube size from 1 .sA to 0.65A allows more accurate predictions to be made because positions of atoms in test compounds are more accurately defined. The presence or absence (coded with *) of a hydrogen on oxygen does not significantly Influence predictive value of the models. Thus, model col 3 can confidently be used to construct the shape of the substrate binding domain for HLADH and is probably the best model format for other dehydrogenases.
526 Table 10. Prediction of acceptance by HLADH using cubic section models with 1.3A or 0.65A cube sizes with the origin at center of the center cube, with and without added hydrogens. Average priority values, unknown and forbidden sites. P(expt) co13 C O 0 6 5 C 0 1 3 * C O 0 6 5 * co13h )o065h Test molecule OH
6."' 32.6
11.2,0,0 10.8,0.0
11.3.0,0 10.9.0,0
11.1.0,0
10.9,1.0
9.6,0.1
9.6,0,1
9.5.0.2
10.8.0,2
OH OH 0
12.4 '
10.4,0.1
10.4.0.1
10.5.0.0 10.8,0.0
10.5,0,0 10.9,0.0
10.9,0.0
11.2,0,0
9.7.0.0
9.6.0.0
9.5,0,0
9.4,0,3
11.5.0.0 10.1,1.0
10.2.2,0
10.1.2,0
OH OH 0
9.1.0.1
9.0.0.1
ccrH
"
OH
^
3.0
OH
11.4.0.0 10.6,1.0
0
7.7,0,3
7.9.0.3
7.5,0.3
7.6,0,3
8.5.0,5
7.1.0,8
0
7.2.0.4
5.4.0.4
6.9.0,4
5.0.0.4
8.7,0,5
5.5,0.11
0
7.2,0.3
5.4.0.3
6.9,0,3
5.0.0.3
8.1,0,5
7.5,0,9
33.6
8.2.0.0
6.2.1.0
8.0,0.0
5.8.1.0
6.3.3.0
7.2.6.0
4.1
8.5.0.0
6.6.2.0
8.3.0.0
6.2.2,0
7.3.3.0
7.5.6,0
0
9.6,0.2
7.0.1.1
9.5.0.2 ' 6.7,0.2
9.3,0,0
5.6,0.3
0
9.1,0.2
5.3.1,1
9.0.0.2
4.8.0.2
10.0.0,0
5.4.0,0
kr""' A>™ 1 1
\
1
h j^H
527
PrexDt^
Test molecule
co13
CO065
C013*
CO065* c o 1 3 h
bo065h
^^fY <*^) •-SOH
1—1
-S;
9.7
10.6.0,0 6.4.3.0
0.3
8.4.0.0
0
0
10.6.0,0 6.1.3.0
8.3.0.0
7.3,5,0
6.0.3.0
8.3.0.0
5.6.3.0
8.2.0,0
4.9,5,0
6.8.0,1
6.1.0.2
6.5.0.1
5.8.0.2
8.2.0,0
5.2,0.5
8.6.0.1
8.1,0,2
8.5,0.1
7.9.0.2
9.1,0,0
6.2.0.6
^
^
1^ 6.6.8.
Comparison of Present Models with X-Ray Crystallographic
Structure The X-ray crystallographic structure of the ternary complex, HLADH-NADH-dlmethyl sulfoxide, DIVISO, has been determined to 2.9 A.34 The crystallographic co-ordinates available from the Brookhaven protein data bank were entered into MACROMODEL and the amino acid residues surrounding the active site identified. Using MACROMODEL and ENZYME the substrate binding domain of HLADH was mapped as a cubic section model. The model obtained from this approach were compared with model col 3 obtained by substrate surrogate overlay. In the comparative study X-ray crystallographic data extending 15 A around the catalytic zinc atom was employed. Using MACROMODEL the sulphur of DMSO in the X-ray structure of the HLADH-NADH-DMSO complex was changed to a carbon reformulating this component as a 2-propanoxy anion with the carbon-oxyanion bond along the previous S-O axis and the carbon-hydrogen bond along to sulphur-lone pair axis. This alcohol structure positioned so that the oxyanion was located 2.2 A from the zinc. Distances between the substrate atoms and the HLADH-NADH residue atoms were determined using MACROMODEL. Table 11 shows the distances between the oxyanion (1), central carbon (1) and the neighbouring HLADH-NADH atoms within 4 A In this complex compared to similar distances for the HLADH-NADH-DMSO and
528 HLADH-NAD-p-bromobenzyl alcohol complexes. The locations of oxygen (1) and carbon (1) In the constructed 2-propanoxy anion-NADH-HLADH complex compared well with analogous distances in the other complexes. The distance from oxyanion (1) to the side chain atoms of HLADH differ only 0-0.5 A between the 2-propanoxyHLADH-NADH and DMSO-HLADH-NADH complexes and 0-0.6 A between the 2propanoxy-HLADH-NADH and PBBA-HLADH-NADH complexes. The location of
Table 11.
Interactions of three ternary complexes of HLADH and NADH. For the pbromobenzyl alcohol complex both observed and productive distances have been given 36, HIadh-Nadh ours HIadh-Nad1 -Dmso(A) Pbba(A) atom residue atom (A) 01
Zn Cys-46 Ser-48 His-67
Cys-174 CoE
C1
Zn Ser-48 His-67
SG OG CB CE-1 CD-2 NE-2 SG C4N C5N C6N
2.2 3.7 2.7
2.2
2.2 (2.2P)
3.6
3.4 (3.5p)
3.7 3.3 3.2
3.2 3.7 3.9
3.3 (3.4p) 3.8 (3.8p)
2.6 3.4
3.1
3.2 (3.2p)
3.6 3.0 3.2 3.8 2.9 3.3
3.5 (3.5p) 3.6 (3.5p) 3.4 (3.5p) 3.9(3.8p) 3.0 (3.1p) 3.4 (3.7p)
3.8 5.0 4.4
3.4
4.9
3.6 (3.6p)
3.6 OG CB CE-1 NE-2
'
3.7
CD-2 Phe-93
CE-2
3.5
4.2
Leu-141
CD-I
3.5
4.3
CZ
3.5
4.1
3.5 3.1
(3.5p)
Cys-174
SG
3.4
(3.6p)
CoE
C3N
3.2
(3.6p)
C4N C5N
5.1
2.4
(3.4p)
3.2
(3.7p)
1
529 carbon (1) differs 0.6-0.8 A In the case of DMSO-HLADH-NADH (except CN4 of NADH where a 2.7 A difference was obtained) and 0.1-1.3 A in the case of the PBBAHLADH-NADH complex from 2-propanoxy-HLADH-NADH complex. The embedded 2-propanoxy group the HLADH-NADH-2-propanoxy complex was oriented as previously described substrate surrogates using MACROMODEL Carbon (1),a-hydrogen and oxygen (1) of the 2-propanoxy anion were used to align the complex, other atoms of this product surrogate were deleted. The MACROMODEL. data file thus created was transferred to ENZYME using MMFORMAT. The complex was aligned as previously described for substrate surrogates and a cubic section model was created with a priority value of zero for all occupied cubes. The coordinates of cubes and atom types of cubes in this model were obtained by using PREDICT-and PRINT CUBEFILE-opWons. It contains 24 layers from-13 to 10. A printed version is available from the authors. We next determined the location in the cubic sector model of those residues lining the substrate binding domain of HLADH. Using the ANALYZE and DISPLAY modes of MACROMODEL (FINDNAME RESIDUE -options, MOLECULE, SET 1 and MONO -options) the residues near the active site pocket were identified and marked. Each marked residue (e.g. Phe-93) was separated from other residues using the DELETE ATOM / BOND command. Each separated residue was connected to the surrogate product to create a rigid complex as needed in the alignment process of ENZYME. Each of the files created by these operations was stored individually. The following residue files were formed: His-67, Cys-174, Cys-46, Leu-57, Ser-48, Phe-93, His-51, Leu-141, Met-306. Phe-110. lle-318, Pro-295, Val-294, Thr-94, Leu-92, Pro-91, Pro-95, Asp-49 and Val-52. These residue files were transferred to ENZYME, using MMFORMAT. The DISPLAY CUBEFILE option was used to determine the position of each residue within the cubic section model generated by ENZYME (shown as white circles each occupied cube). The co-ordinates of atoms in each residue were obtained from this process by creation of individual files for each residue and each of these was converted to a cubic section model. Addition of the residue cubic section models gave a cubic sector model of the substrate binding domain consisting only of the residues lining the this pocket. Model 0013 and the model constructed with all hydrogens in substrate surrogates (co13h) were compared with the cubic section model derived from display of the cubes occupied by residues of HLADH lining the substrate binding domain. Using
530 COMPARE the X-ray based model was overlaid with the substrate surrogate derived models.
Forbidden cubes in c o 1 3 . c o 1 3 h and the X-ray derived model which
overlaid were identified and displayed (Figure 20). The co-ordinates of each cube and atom types in each cube were also identified. This allowed location of the residues lining the substrate binding domain that interfered with substrate binding. Only layers between -3 to 4 have significant overlap between the models. We commenced analysis of layers at layer -3, then proceeded to layer -2 and so on. The coenzyme, NADH was located in the front layers. The forbidden cubes in layers -3, -2 and -1 are close to the coenzyme and residues Phe-93, lle-318, Cys-174 and His-67. Residues Leu-309, Val-309 and Thr-94 with attached hydrogens may effect layer 0. The coenzyme and Phe-93 are the closest to the cubes -1H, -1K and -10 In model c o l 3 . For some bridged substrates (30, 31) these cubes are allowed in this model. In layer 0 the residues Leu-309, lle-318 and Val-294 affect the cubes above OM to OR and may be In the boundary area. Residues Phe-93, lle-318 and possibly Hls-67 affect the forbidden cubes on the right side of model co13. Val-294, Ser-48 and coenzyme are the residues nearest to the forbidden left edge. According to the Xray derived cubic section model the left side of the substrate binding domain (in layers 1-4) Is more open because Val-294 and Leu-309 can change orientation. In layer 1 Phe-93, Thr-94, His-67 and lle-318 affect the forbidden right side and lle-318 affects the cubes above ON to OQ in model co13. Ser-48 and His-51 are quite close to the left edge of co13. This edge is assumed to be the boundary area. It is forbidden for some substrates and allowed for others. In layer 2 the right edge of co13 Is affected by Hls-67, Phe-93, Leu-116, Leu-141, lle-318 and Phe-110. The Van der Waals interactions from other layers (1 and 3) have an especially important role in layer 2 definition. His-51 and Ser-48 may affect the left edge, an area that seems to be allowed for substrates with phenyl groups. The upper part of the left side of co13 is the most open with Val-294, Leu-309 and Pro-295 as the nearest residues. The same residues affect layers 2 and 3. Whereas residues Leu-57 and Phe-140 affect layer 4. The cubes in the upper edge of the left side of layers 2 and 3 are defined from larger substrate surrogates. They may affect orientations of the side chains of Val-294 and Leu-309. Leu-116 and Met-306 have also been suggested to change their side chain conformations with more bulky substrates (38) however, Met-306 Is quite deep In layers 3 and 4 In present X-ray based model. Comparison of the shapes of the substrate binding domain derived from these two approaches provides a good view of the domain.
531 Jones manually derived positions of amino acid residues in the apoenzyme structure and some ternary complexes published before 1977. Figure 21 shows a comparison between the present results with the earlier definition. The boundary region of model (0013) has also been assigned in the Figure. Ser-48, Phe-93 and lle-318 are in the same location while Leu-57 is in layers 3, 4 and 5 In our X-ray derived model. Using the present method positions of residues were positioned more accurately.
(H)
(C)
(N)
(N)
nil [TJ G
(Ip) CoE (C,H)
(0)
(Ip)
CoE
CoE
CoE
A
0
P
Q
Tn
H
1(H) J
K
L
B
CoE D C CoE^)
E
F
uE
uF
uA(C •uB(C) uC(K) uD |coE CoE CoE
(C)
(S) C174 (H) (C) (H) C174 a? A
(C)
(H) C-46
layer -3 (C)
CI 74 stoiTi type (C) and the amino acid residue, Cys-174 forbidden allowed for some 3-alkylcyclohexanols or heterocyclic bicyclic alcohols, usually forbidden allowed for 4-alkylcyclohexanols, usually unknown Figure 20. The overlaid layers with X-ray based model and present models oo13 (upper) and oo13h (lower).
532
(0) 1-318
(0)
1-318 (H) (H) CoE 1-318 (C)
(H)
(0)
(H) CoE
F M ^ ^ G (C) CoE BCoB
[uA
(C) CoE CoE
(N) CoE
acog adoB J
"(C) '(C) CoE CoE C D B
uB
(H)
(H)
o u C ^ uD
1'1 K 1
E
uE
""(Hi P-93J uF
(H)
|p-93 (H) CoE (0)
(H)
(C)
(H)
C-46 (S)
C-46 (H) (C) C-46 C-46
layer - 2
(H)
(H)
533
RcJo
pcoTl
1-318 (C) h-318 1 (C)
pi (H) CoE
G
[0 1 0.02 J H 1 1 34
C
A
(Ip) CoE
(C) ! uA CoE
(0.1 P: qoE
TH
[N
NJB
K 1 L 4
p 55 ^ ^
uC ^ uD 45 11
uE
uF
(C,C) |P-93 (H) P-93
10 9 (H) H-67
(H)
(c)
(C)|
(N) H-67
(C) (C,H)i (N)
(c) 1 (H)
(0)1 H-67
C-46 C-46
layer -1 (C,H) 1-318
(0)
(C) 1-318 (C)
(H) CoE
[G A
(Ip) CoE
0 2 P 2^ ^ R 3 17 JHIC 1 9 J 6 K 3 2 4 4 16 D 55 E 3 B 2 c 2
(C) CoE
Ko.ip;
12
^ ^ u C
^ uD 1 uE
44 11
10 8
JP93| KC^ P-93
WPS
(H)
(H) H-67
(C) (C.H)
mi
1 I
(C)
1
(N) H-67
(N)
(C) (H) C-46 C-46
(0) |H-67
534
layer 0
535
layer 1
536
537
ric)
(H)
rw
M-306 M-306
(H)
P-110
P-295
p-no
(H)
(H) V-294
[ M ^ pT"
PTHT \Q
0
[R
1
L-116
H G 61'^ V-294 H-294 2 A "Z B 'A 61 11 31 (H)
uA
c
ZTD 1
•(d) P-93
A^yV.
uB
rTc)
u E ( H ^ ^ a (H) (C) L-141 (H)
1 28
H-51
1 28 , (H)
(0)
H-51 (N)j
S-48 (H)|
H-5l!
A-49
L-141
(Ip.O)
{H)| P-14(^
layer 3 (C)\
(C)
M-306 M-306
p-no (H)
(H) (H)
p-no
P-29S 1 (C) |L-n6
11 121 (H) V-294
IM
N
\ 0
R
^J»57
(C)
3 4
(H) H-51 (N) H-51
1
1.3 H 4 1
G 5J 4 61^ 61^ 7 1 25 ^ ^ ^ 4 V-294 H-294 4 A 7 B 13 C 5| D 8 E 4 1 121 46 17 16 1 1 8^ 0.6^ 19 10 2 2
H-51
^P
28^ 0,48
"loTJ uF 1 m
p-93
^1
(C) l " 2 16 ^ ^ 0.22 L-141 (H) 1 28 L-141 1 4
(H)
(H)
A-49
P-140
moi
538 IHTI
(H)
(H)
M-306
M-306
p-no
(H) L-116
(H) L-ne
[M
(H)
(H)
N
0
P
1
•
G
H
A
B (H
>oo?
uA
L-57 uB(C) L-57
L-57
lr-57
Q(C) L-ne K
D
"Wl
(H)
"mlflcT 1 L-n 6 1-116 L
F(H)
(H)
L-14l| | L - 1 4 1
^uD
(H)
• 1
(H) L-141 (C)
L-141
H-51
(C) L-141
(C)
(H)
(C,H)
(C,H)
H-51
H-51
P-140
L-141
(H)
(C) 1 ^^^ P-140
H-51 (H) V-52
layer 4 (H)
(H)
(H)
M-306
M-306
p-110
(H) L-116
(H) L-ne
\IA
(H)
(H)
r^
0
H
A
B 1 C 1 3.5 3.0 H,57 uB(C]
uA
L-57|
P
K
1
^ ^ ^^^ ^^^
L
F(H)
(H)
L-14l| L-141 uF
(H)
1 57K
L-141 (C)
(H)
(C)
L-141 L-141
H-51 (C)
(H)
1 L-116 L-116| [1-116
G
L-57
(HTI
(H)
H-51 H-51
(C.H)
(C,H)
P-140
L-141
(C)
(C)
H-51
p-140 (H) V-52
(H)
539 Jones model 309
[306
3od sial am
p!95
cW\ ^
294 294 294
layer 2
Ju r
mo
>^;
294
110
51
1''
^
51
1411 48
48
I 93
1141
48
HO]
48
110^
309
309
bisj
295
318 318
i94^H
294
layer 1
94
294 93 MM
k^
1
1
51
48
309
48
no^
bisl
309
318
309
3ji
COEI
iK
c^
S — < ,—1
• H ESil JS? mm w^m mm ^mm >S$^ layer 0
u
93
CoE
|H-5J
CoE
^
M
—<
MP
48 48
-^^^ . . • torDidden [ ^ BMhJ or limited
93
93
48 67
, „ „. allowed boundary \ ^ in some KX^ ^^^^«
94
^Mji"
[2*94^^
294
^.^.^^
Ur
67
^
1
forbidden
Figure 21. Comparison of Jones map of amino acid residues with present definition.
540 6.7.
CONCLUSIONS
The present computer-based substrate surrogate overlay method gave a refined cubic section model for the substrate binding cavity of HLADH. Allowed and forbidden regions were accurately defined using the origin in the corner of a cube and a cube size 1.3 A or 0.65A (model co13 and co065). Boundary regions (Figure 21) which can be either allowed or forbidden were defined. The average acceptance value in these regions was usually 2 or lower. Low average priority values for some cubes (Figure 16: OG, ON, OQ, OK, 1 G, 1 K, l u E , 2K, and 2E) are due to the inclusion of bulky substrate surrogates with low priority values in construction of the model. These are assumed to change the orientation of the side chains of amino acid residues lining the substrate binding domain. Reducing the cube size to 0.65A (co065) from l.sA (co13) primarily affects cubes OH, OK, ON, OQ, 1K, 1H, and IN in the 1.3A model by defining those parts of these boundary cubes that are forbidden or allowed. The cubic section models, co13 and co065, proved to be accurate in predicting acceptance of substrates and the stereochemical course of HLADH mediated reductions. The average priority value for all cubes, number of forbidden sites and number of low priority cubes occupied by atoms in a possible alcohol are used to estimate the acceptability of the corresponding ketone by HLADH. The prediction method developed can be applied to other enzymes (e.g. other dehydrogenases, esterases, or lipases) if kinetic and specificity data is available. Comparison of the X-ray based cubic section model with the substrate surrogate derived cubic section model gave a view of the amino acid residues around the substrate binding domain (Figure 21). Forbidden and boundary regions defined by the substrate surrogate overlay method are near the side chains amino acid residues. Substrate interactions with surface of the amino acid residues could be estimated by molecular mechanics (MACROMODEL). The close resemblance of the present models, developed with rather sophisticated computer techniques, to the original Jones model, developed with hand held stick models, is a testimony to the ingenuity of the Jones group. 6.8.
ACKNOWLEDGEMENTS The authors acknowledge the financial support of Suomen Kulttuurirahasto,
Neste Oy and Kemira Oy of Finland and the Natural Sciences and Engineering
541 Research Council of Canada. As well the authors thank Mr. L. Corey and Ms. Sharon Cyprik for development of the ENZYME and COMPARE programs and Clint Surry and Dr. All Mohammed for help in preparation of the manuscript. M.A. sincerely thanks Professor H. Krieger for his support and encouragement of her studies in Canada.
6.9. REFERENCES 1. E. L Smith, R. L Hill, I. R. Lehman, R. J. Lefkowitz, P. Handler & A. White, in: R. S. Laufer, E. Warren & D. Mclvor (Eds), Principles of Biochemistry: general aspects, R. P. Donnelley & Sons Company, 1983. 2. C.-H. Wong, Science, 244 (1989) 1145. 3. A. Akiyama, M. D. Bednarski, M. J. Kim, E. S. Simon, H. Waldmann & G. M. Whitesides, Chemtech, (October 1988) 627. 4. J. B. Jones, Tetrahedron, 42 (1986) 3351. 5. G. M. Whitesides & C.-H. Wong, Angewandte Chemie, 24 (1985) 617. 6. M. Schneider, Enzymes as Catalysts in Organic Synthesis, Reidel, Dordrecht,1986. 7. J. B. Jones, in: P. Dunnill, A. Wiseman and N. Blakeborough (Eds), Enzymes and Nonenzymic Catalysis, Horwood/Wiley, Chichester/New York,1980. 8. J. B. Jones, C. J. Sih & D. Perlman, Applications of Biochemical Systems in Organic Chemistry, John Wiley & Sons, inc., 1976. 9. J. Retey & J. A. Robinson, Stereospeciflcity in Organic Chemistry and Enzymology, Verlag Chemie, Weinhelm/Deerfield Beach, Florida/Basel,1982. 10. A. Fischli, in: R. Scheffold (Ed.), Modern Synthetic Methods, Vol. 2, SalleSauerlander, Frankfurt, 1980. 11. J. B. Jones, in: J. R. Morrison (Ed.), Asymmetric Synthesis, Vol. 5, Academic Press, New York, 1980. 12. P. Sonnet, Chemtech, (Fehruary 1988) 94. 13. Enzyme Nomenclature, Elsevier, Amsterdam, 1973.
542 14. K. Martinek & J. J. Semenov, Appl. Biochem., 3 (1981) 93. 15. H. Theorell, in: R. G. Thurman, T. Yonetani, J. R. Williamson & B. Chance (Eds.), Alcohol and Aldehyde Metabolizing Systems, Academic Press, Inc.,1974. 16. C.-l. Branden, H. Jornvall, H. EkIund & B. Furugen, in: P.D. Boyer (Ed.), The Enzymes, Vol. XI, Part A, Academic Press, New York, 1975. 17. H. EkIund & C.-l. Branden, in: T. G. Spiro (Ed.), Zinc Enzymes, John Wiley & Sons, Inc., 1983. 18. H. EkIund & C.-l. Branden, in: F. A. Jurnak and A. McPherson (Eds), Biological Macromolecules and Assembles, Vol. 3, John Wiley & Sons, Inc., 1983. 19. V. C. Sekhar & B. V. Blapp, Biochemistry, 27 (1988) 5082. 20. J. P. Klinman, CRC Critical Reviews in Biochemistry, 10 (1981) 39. 21. J. B. Jones, Enzyme Engineering, 6 (1982)107. 22. A. I. Irwin & J. B. Jones, J. Am. Chem. Soc, 98 (1976) 8476. 23. D. R. Dodds & J. B. Jones, J. Chem. Soc, Chem. Commun., (1982).1080; J. Am. Chem. Soc, 110(1988)577. 24. A. J. Irwin & J. B. Jones, J. Am. Chem. Soc. 99 (1976),1625. 25. I. J. Jacovac, G. Ng, K. P. Lok, & J. B. Jones, J. Chem. Soc, Chem. Commun., (1980) 515. 26. J. Davies & J. B. Jones, J. Am. Chem. Soc, 101 (1979) 5405. 27. M. A. Findeis & G. M. Whitesides, Ann. Rep. Med. Chem., 19 (1984) 263. 28. J. B. Jones & K. E. Taylor, Can. J. Chem., 54 (1976) 2969. 29. C.-H. Wong & G. M. Whitesides, J. Amer. Chem. Soc, 103 (1981) 4890. 30. W. H. Baricos, R. P. Chambers & N. Cohen. Anal. Lett., 9 (1976) 257. 31. A. R. Fersht, in: Freeman (Ed.), Enzyme Structure and Mechanism, New York, 1985.
543 32. R. W. Hay, Bio-inorganic Chemistry, Ellis Norwood, Halsted Press, 1989. 33. H. Ekiund, B. Nordstrom, E. Zeppezauer, G. Soderlund, I. Ohisson, T. Boiwe, B.-O. Soderberg, O. Tapia, C.-l. Branden, and A. Akerson, J. Mol. Biol., 102 (1976) 27. 34. H. Ekiund, J.-P. Samama, L Wallen, C.-l. Brand6n, A. Akeson & T. A. Jones, J. Mol. Biol., 146(1981)561. 35. H. Ekiund, J.-P. Samama & L Wallen, Biochem., 21 (1982) 4858. 36. H. Ekiund, B. V. Plapp, J.-P. Samama & C.-l. BrSnd^n, The J. of Biol. Chem., 257 (1982)14349. 37. E. Cedergren-Zeppezauer, J.-P. Samama & H. Ekiund, Biochem., 21 (1982) 4895. 38. E. Horjales, H. Ekiund & C.-l. Brarlden, J. Mol. Biol., 197 (1987) 685. 39. H. Jornvall, Eur. J. Biochem., 16 (1970) 25. 40. H. Ekiund, Biochem. Soc. Trans., 17 (1989) 293. 41. V. Prelog, Pure Appl. Chem., 9 (1964) 119. 42. A. J. Irwin & J. B. Jones, J. Am. Chem. Soc, 99 (1977) 556. 43. A. J. iHA^in & J. B. Jones, J. Am. Chem. Soc, 99 (1977) 1625. 44. E. Horjales & C.-l. Branden, J. Biol. Chem,, 260 (1985) 15445. 45. J. B. Jones & I. J. Jacovac, Can. J. Chem., 60 (1982) 19. 46. J. M. H. Graves, A. Clark & H. J. Ringold, Biochem., 4 (1965) 2655. 47. M. Nakazaki, H. Chlkamatsu, K. Naemura, Y. Sasaki & T. FujIjI, J. Chem. Soc, Chem. Commun., (1980) 626. 48. H. Duller & C.-l. Branden, Bioorg. Chem., 10 (1981) 1. 49. G. L Lemiere, T. A. Van Osselaer, J. A. Lepolvre & F. C. Alderweireldt, J. Chem. Soc, Perkin Trans II, (1982) 1123.
544 50. R. A. Johnson, in: W. S. Trahanovsky (Ed.), In Oxidation in Organic Chemistry, Part C. Academic Press, New York, 1978. 51. M. Nakazaki, H. Chikarnatsu, K. Naemura & M. Asao, J. Org. Chem., 45 (1980) 4432. 52. J. J. Willaert, G. L. Lemiere, L. A. Joris, J. A. Lepoivre & F. C. Alderweireldt, Bioorganic Chemistry, 16 (1988) 223. 53. J. B. Jones & T. Takemura, Can. J. Chem., 60 (1982) 2950. 54. J. A. Haslegrave & J. B. Jones, J. Am. Chem. Soc, 104 (1982) 4666. 55. T. Takemura & J. B. Jones, J. Org. Chem., 48 (1983) 791. 56. M. Nakazaki, H. Chikamatsu & Y. Sasaki, J. Org. Chem., 48 (1983) 2506. 57. M. Nakazaki, H. Ckikamatsu & Y. Sasaki, J. Org. Chem., 48 (1983) 4337. 58. M. Nakazaki, H. Chikamatsu, K. Naemura, T. Suzuki, M. Iwasaki, Y. Sasaki & T. Fujiji. J. Org. Chem., 46 (1981) 2726. 59. T. A. Van Osselaer, G. L Lemiere, J. A. Lepoivre & F. C. Alderweireldt, Bull. Soc. Chim. Belg., 89(1980)389. 60. T. A. Van Osselaer, G. L, Lemiere, J. A. Lepoivre & F.C. Alderweireldt, Bull. Soc. Chim. Belg., 89(1980) 133. 61. J. Van Luppen, J. A. Lepoivre, G. L. Lemiere & F. C. Alderweireldt, Heterocycles, 22 (1984) 749 62. A. R. Krawczyk & J. B. Jones, J. Org. Chem., 54 (1989)1795. 63. L K. P. Lam, I. A. Gair & J. B. Jones, J. Org. Chem, 53 (1988) 1611. 64. C.-S. Chen, Y. Fujinloto, G. Girdaukas & C. J. Sih, J. Am. Chem. Soc, 104 (1982) 7294.
545 6.10.
Appendices
Appendix 1 : MACROIVIODEL Description After connecting to the VAX and setting the correct sub directory (e.g. set default [site]), the program MACROMODEL is started by typing RMMOD. When the display is shown on the screen, active buttons are shown in green. The first mouse key is shown as the pick key. The second mouse key acts the same for ail buttons except for the DRAW button. The third mouse key will display the help screen for that button. The molecule on the screen will be drawn by using the DRAW button active. The first key draws connecting bonds to subsequent points whereas the second key restarts the drawing of bonds. The hydrogens are added by choosing the H ADD button three times. Non carbons are replaced by choosing the appropriate symbol and picking the atom to replace. Functional groups can also be appended in this manner. The structure is then minimized in energy by selecting the ENERGY mode (choose MM2 force field) and picking start.
Constraints are added by selecting the
CONSTRAINT submode and selecting the appropriate choice of constraint (CATM, CDIS, CBOND, CTOR) and choosing the atoms involved. To orient the structure, pick the ANALYZE mode and pick ALIGN X, ALIGN Y, ALIGN Z, ROTX, ROT / a n d ROTZ as needed. The aline buttons need two atoms picked after the command to align on the axis and the rotate buttons need a typed input on the number of degrees to rotate on that axis. The structure is stored by picking the WRITE button an supplying a filename (the extension defaults to .DAT) and give a blank line for sequence structure number. When finished, exit the program by picking the STOP button and confirming that you wish to exit. Answer "Y" to the question for deleting the log file.
546 Appendix 2:
ENZYME and COMPARE Documentation
To execute the ENZYME program, type RUN ENZYME and the graphics screen with a menu is displayed as in Figure 12. The choice of options are: READ CUBEFILE, READ DATA, ADD TO CUBE FILE, REMOVE FROM CUBEFILE, ALIGNMENT, ROTATE ON X AXIS, ROTATE ON Y AXIS, ROTATE ON Z AXIS, PREDICT PRIORITY FOR MOLECULE, DISPLAY CUBEFILE, ROTATE ABOUT BOND, PRINT CUBEFILE, SAVE CUBEFILE, EXIT, CHANGE CUBE SIZE, CHANGE ORIGIN IN CUBE, and DISPLAY CUBE WITH DIFFERENT PRECISION. The desired option is chosen by depressing a mouse button while pointing the cursor at the option and the exact operation to be performed is dependent on which of three buttons was pressed. 1. Read cube file {READ CF): This option asks for the cube file to read; the extension .cf will be added to the file name. 2. Read data (READ DATA): This option reads in the data generated by MACROMODEL program (the extension must be '. dat' and the file must be in ASCII format which is made by running MMFORMATor\ the file made by MACROMODEL and supplying the filename) and asks for the necessary information to determine its priority. The first button will direct the program to follow this section by the alignment section; the second button directs the program to return to the menu. 3. Add to cube file {ADD TO CF): This option adds the molecule to the cube file; It first checks to see if the molecule has already been aliened. 4. Remove from cube file {REMOVE CF): This option removes the priority from the cubes that are occupied by the molecule. It does not check the cube file for the presence of the molecule since it is impossible to confirm the previous addition of the molecule. The molecule must have the same priority ranking as it had when it was added. 5. Alignment {ALIGNMENT): The option aligns the molecule in the reference axes with one of the atoms on the origin with a relative co-ordinate of (0, 0, 0). It will ask for the atom to be placed on the origin, the atom to be on an axis which is chosen by one of the buttons, and the atom to be placed in a plane which is chosen by one on the buttons. When choosing this section with the second button, the atom chosen will be translated to the origin and the alignment of the rest molecule is not done. 6. Rotate on x axis {ROTATE ON X), rotate on y axis {ROTATE ON Y), rotate on z axis {ROTATE ON Z): These options will rotate the molecule on the specified axis by an
547 amount which is typed in. Depression of the second button will rotate the molecule on the axis by 180**. All rotations are positive in the right handed co-ordinate system. 7. Predict priority for molecule (PREDICT): This option determines the priority of the molecule based on the information in the cube file. The second button will write the information, and how each atom fits, in a file with the same name as the molecule but with the extension '.pre*. 8. Display cube file (DISPLAY CF): This option displays the cube file by displaying each layer of the cube file starting from the near side. It will either display the cube file with 3 colors or 6 colors depending on the value shown for the last option. The displayed layers will be limited (from -5 to +10 inclusive) if the first button is pressed and will be unlimited if the second button is pressed. If a molecule shown on the screen, then circles within the cubes are drawn to indicate which cubes the molecule occupies. 9. Rotate about bonds (ROTATE BOND): This option rotates a group of atoms the specified bond and returns the orientation with the best fit for the cube file; it does not add the molecule to the cube file. 10. Print cube file (PRINT CF): This creates a file which contains the cube coordinates and the average priority for the cube. It also includes the number of atoms in the cube. 11. Save cube file (SAVE CF): This saves the cube file. 12. Exit (EXIT): This option also saves the cube file if it has been changed and exits the program. 13. Change cube size (CF number): This option allows the size of the cube to be changed. It does not recalculate the cube file to reflect a change in size of the cube so a different cube file should always be used with each cube size. If you wish a size different from the default, specify the change in size before doing any operations on the cube file so the cube file will not contain garbage. 14. Change origin in cube (CORNER or CENTER): This option moves the origin of the axes to the corner of the cube or the center of the cube. The place on the origin is reflected by location of the origin, specify the change before doing any operations on the cube file. 15. Display cube with different precision (DISCOL number): This option determines if the cube file Is displayed In 3 or 6 colors with different ranges.
548 To Start to execution of COMPARE, type RUN COMPARE after exiting ENZYME program. Then choose option READ CUBEFILE and give the name of first cube file and also the name of second cube file. After it choose option DISPLAY CUBEFILE and it gives the overlaid picture of these cube files. The overlaid cubes can be seen with different colors: forbidden in first cube file (black), forbidden in both cube files (red), conflict in first cube file (blue), conflict in second cube file (yellow), and allowed in both cube files (green). In the end of the program it gives the number of conflicts in both cube files.