Serine hydrolase-phosphyl ester interactions: Molecular modeling

Journal of Molecular Structure (Theochern), 170 (1988) 159-169 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

SERINE HYDROLASE-PHOSPHYL MOLECULAR MODELING

159

ESTER INTERACTIONS:

ILDIKO M. KOVACH The University of Kansas, Center for Biomedical Research, 2099 Constant Avenue, Lawrence, KS 66045 (U.S.A.) (Received 20 September 1987)

ABSTRACT Energies of interactions for trypsin adducts phosphorylated at the active-site Ser-195, seryl2propylphosphate anion, with and without a proton on His-57 at the active-site, and seryl diisopropylphosphate with the protonated His-57 were calculated with the molecular mechanics program YETI. The total energy of stabilization is similar for the adducts with protonated His-57, 20-60 kcal mol-’ lower than for the unprotonated adduct, and dependent on the choice of the dielectric parameter. In all three adducts, two H-bonding interactions were calculated in the oxyanion hole to give - 3.5 to - 4.5 kcal mol- ’ mostly dependent on the dielectric parameter. Additional stabilizing interactions between the protonated His-57 and the phosphate anion are -2 kcal mol-’ due to H-bonding and at least -5 kcal mol-’ due to electrostatic forces. For this adduct, the energies of interactions at the active-site total to < - 11 kcal mol-‘. The equilibrium energy of formation of diethylphosphate adducts of acetylcholmesterase are also in the neighborhood of - 10 kcal mol-’ more similar to the pro&mated than the unprotonated adduct. Stereoscopic visualization of the structures after final refinement was generated with the use of FRODO.

INTRODUCTION

The toxic effects of organophosphorus (OP) compounds stem from their nearly irreversible inhibition of the catalytic action of serine hydrolases: acetylcholinesterase (AChE) and the serine proteases [l-2]. Through certain characteristic interactions between the compounds and the hydrolases, the enzyme’s catalytic apparatus is mobilized to effect rapid phosphylation, a process that is similar to acylation in the analogous reactions with natural substrates of the enzymes (Scheme 1). R,R,POX+EOH+R,R,POE+X-

(1)

While the substrates form transient intermediates, the enzyme-OP adducts are stabilized by interactions between the active-site residues of the enzyme and the ligands around the phosphorus atom. The consequence is that, unlike 0166-1280/88/$03.50

0 1988 Elsevier Science Publishers B.V.

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with the substrates, the phosphyl group does not readily hydrolyze off the enzyme, the reason for which is, at best, poorly understood. An increasing availability of structural details, by X-ray and NMR techniques, for serine protease catalysis and inhibition, could shed some light on the types of forces involved in stabilization of the phosphylated serine hydrolases. In the wake of current discoveries [3] on the molecular dynamics of catalysis by serine proteases and of their inactivation by diisopropyl phosphorofluoridate one can perceive a more holistic explanation of the formation of the surprisingly stable phosphyl-serine hydrolase adducts. Dynamics

A combination of two structural factors has a profound effect on the dynamics of serine protease catalysis [ 31. The concurrence of the formation of the tetrahedral intermediate and the proton transfer from Ser-195 to the ENA of His-57 are necessary for a slight movement, a tilt <30”, of the His-57 ring away from the Asp-102 [ 41. This results in the splitting of the Asp-His H-bond and the approach of the leaving group by the HeNA of the His-57 [ 31. By this movement of the His-57, the HENA is correctly oriented for general acid catalysis of the departure of a peptide (and, supposedly, other poor) leaving group(s). It is not clear what the orientation of the HeNA of His-57 becomes without the formation of the evolutionarily anticipated ion-pair in which the negative charge is localized in tetrahedral geometry and anchored by two Hbonds in the oxyanion hole, but even if the anticipated movement of the His57 can be effected by the formation of pentavalent phosphyl-enzyme species, the leaving group of this species will also be in the wrong position for the His57 to assist in its departure. The leaving group is expected to be in the axial position, 180” from the Ser-190-O-P bond (A), or in the equatorial position, at 90” from the Ser-195-0-P bond [5], compared to the tetrahedral angle at 109’ for the substrate reaction (B). Under these circumstances the His-57 probably could not promote departure of the leaving group from the pentavalent phosphorus adduct. OE

I

c

Rh.

/ X

A

‘Q

B

For the most reactive OPs, departure still takes place efficiently, since good leaving groups are involved. Alternatively, catalysis by some other residue or water, possibly needed for the departure of F-, could be involved. Thus, phosphylation of serine hydrolases can take place with general base catalysis of the attack of Sr-195 [6], presumably, through the formation of a pentacovalent

161

species with either concerted or time-shifted departure of the leaving group, but without catalytic participation of the His-57 in the latter step. The phosphyl-enzyme This stable intermediate, however, geometrically resembles the tetrahedral intermediate in the deacylation of serine hydrolases. If so, the forces that stabilize the tetrahedral adduct can act on the phosphylated enzyme; that is, the charge density distribution of this phosphyl-enzyme resembles that of the tetrahedral intermediate. As suggested by the X-ray structure of monoisopropyltrypsin [7], two stable H-bonds are formed between the free 0 on the phosphorus and the NHs of the Ser-195 and Gly-192 in the oxyanion hole. This fixation must confer a fairly stringent spatial arrangement upon the rest of the molecule, locating one ligand on the ,phosphorus within the sphere of the HENA of His-57 for H-bonding. If the H1s-57 ring had not previously moved (during proton transfer from Ser-195), it certainly could at this late stage, when tetrahedrality is attained in the stable phosphyl-adduct. This phase shift for the conformational adaptation to the requirement for protonation of His57, compared to the sequence in acylation has a serious consequence. A devastating additional H-bond can form between an electron rich (alkoxy) ligand of the posphorus if it gets within the proper distance from the HENA of the His-57. The third stabilizing H-bond becomes even more counterproductive for catalysis when it promotes departure of the alkyl ligand attached to the H-bonded 0, thereby generating a negatively charged adduct [ 71: a process called “aging” [ 2 1. The negatively charged enzyme-adduct adheres even more strongly to the active-site region of the enzyme by the same type of electrostatic (H-bond) forces. The character of non-bonded interactions that stabilize the phosphyl-serine protease adducts compared to those of the “aged” adducts with and without the second hydrogen and positive charge on His-57, has been the subject of studies reported herein. RESULTS

Molecular mechanics calculations, with program YET1 [ 81, were carried out for covalent adducts formed betwen diisopropyl fluorophosphate (DFP) and trypsin [ 71. Fifty two amino acid residues, twelve water molecules, and the inhibitor (506-510 atoms in total) were included in a sphere of a 10 A radius around the y0 of Ser-195 at the active-site. X-ray crystallographic parameters for trypsin-seryl (Ser-195) monoisopropylphosphate (MIPSE), the “aged” adduct (MIPHIS ) [ 71, were used from the Brookhaven Data Bank (PDB) [ 91. Location of the hydrogens on the His residues were determined by inspection in FRODO [lo]and were assigned by the use of program HYDPOS

162 -303

+310/

014 = Cl3 \ +168 c3

+112

-

-344

c4 \

/

-305 o5

***

H2 - Nl +201

i;+3y

I

-370 \

,;1

-

1081

Cl1

Fig. 1. Connectivity, numbering and atomic partial charges ( X 1000) for MIPSE. TABLE 1 Interaction energies ( - kcal mol-I)

for trypsin adducta, at the active site

Atoms involved

Interacting residues

%Qeof interaction

MIPHIS D=lr

Asp 102-His(p) Asp 102-His(p) Asp 102-His(p) Asp 102-H&(p)

57 57 57 57

HN89 0(2)162 0(2)162.**.HNSS HNA94 0(2)163 O(2)163....HNA94

El HB El HB

Sum of energies Asp 102-His(p) 57 Hip 57-Sub 500 Hip 57-Sub 500

HNA352 HNA352.

OP5008

**.OP5008

D=4r

Sum of energies atP=O Sum of energies at ache site

0 5007....HN298 0 5007....HN5002

D=4r

D=lr

Ddr

2.52 2.32 3.05 2.62

10.13 2.36 17.53 2.10

2.51 2.26 4.08 2.74

10.09 2.32 17.72 1.00

2.54 2.38 4.17 2.05

27.16

10.51

32.12

11.59

31.13

11.14

20.69 1.96

4.98 2.23

Sum of energies Hip 57-Sub 500 Sub 500-Gly 193 Sub 500-Ser 195

D=lr

10.19 2.42 12.82 1.73

El HB

HB HB

DIPHIP

MIPHIP

22.65

7.21

2.30 0.99

2.74 1.90

1.63 1.79

2.58 1.91

2.68 1.76

2.56 1.98

3.29

4.64

3.42

4.49

4.44

4.54

30.45

15.15

58.19

23.29

35.57

15.68

[ 81. Adducts with both the protonated form, Hip-57 (MIPHIP), and its unprotonated form (MIPHIS) bearing the hydrogen on 6N were generated for minimization. energy Trypsin-sexy1 (Ser-195) diisopropylphosphate (DIPSE) with His-57 protonated (DIPHIP) has also been studied. The isopropyl group pointing “up” in DIPSE was built by using the same internal

163

Fig. 2. Stereoscopic view of the active-site of MIPHIS. Labele: Asp-102 y0, His-57 cwN,tN, Gly103 aN, Try-215 /JC, and MIPSE-500 Nl and P.

Fig. 3. Stereoscopic view of the active-site of MIPHIP. The van der Waals’ volume (r= 1 A) of the MIPSE adduct is represented with dots.

coordinates as for the “down” position obtained from the X-ray data for MIPSE. Nonbonded interactions numbered up to 43000 for final refinement with smooth cut-off criteria of 7.5-8.0 A for van der Waals’, 6.5-7.0 A for electrostatic, and 4.5-5.0 A for H-bonding interactions. The convergence criteria were set to 0.025 kcal mol-’ deg-’ for torsional RMS first derivatives, to 0.050 kcal mol-’

164

Fig. 4. Stereoscopic view of the active-site of DIPHIP. The van der Waals’ volume (r= 1 A) of the DIPSE adduct is represented with dots.

deg-l for rotational RMS first derivatives, and to 0.750 kcal mol-’ A-’ for translational RMS first derivatives. The inhibitor covalently bonded to Ser-195, all water molecules included, and the side-chains of those amino acids whose surroundings were defined within the extended active-site were relaxed. Figure 1 shows the connectivity, numbering, and partial charges for MIPSE. The charges indicated at individual atoms are “best values” adopted partially from a calculation with SYBYL [ 111 for the phosphates in NAD and partially from the AMBER library [ 121. TABLE 2 Energy ( - kcal mol-‘) Interacting entities

partition for refined conformations of trypsin adducts MIPHIS

DIPHIP

MIPHIP

D=lr

D=4r

D=lr

D=4r

D=lr

D=4r

Protein-protein Substrate-substrate Protein-substrate Protein-water Substrate-water Water-water

365.95 -2.32 36.37 118.30 1.81 5.13

25857 -2.26 29.98 86.32 1.23 2.27

381.42 -2.25 68.03 120.80 3.96 6.14

263.97 - 2.23 40.36 87.27 1.59 2.49

388.50 -4.02 51.94 125.04 3.21 2.12

264.50 -4.17 38.31 91.82 2.14 1.14

Total

525.24

376.12

584.10

393.44

566.77

393.12

165 TABLE 3 Stabilizing interaction energiesa ( - kcal mol- ’ ) for trypsin adducts Interacting residues Atoms involved

Asp 189-Val17 His 40-GIy 193 Wat 675-His 40 Gly 43-Sub 500 Ala 55-Ser 54 Tyr 94-Wat 614 Thr 229-Asp 102 Wat 637-Ser 139 Trp 141-Wat 616 Wat 639-Gly 142 Asp 189-Ala 221 Ala 221-Asp 189 Asp 189-Wat 657 Wat 687-Asp 189 Asp 189-Wat 659 Wat 659-Asp 189 Cyx 191-Asp 194 Cyx 191-Asp 194 Asp 194-Cyx 191 Wat 616-Gly 193 Gly 197-Asp 194 Wat 637-Asp 194 Gly 196-VaI 213 Thr 229-Ile 212 Ser 214-Wat 664 Wat 626-Ser 214 Wat 671-Ser 217 Wat 671-Lys 224 Wat 661-Val227 Wat 660-Wat 626 Gly 216-Wat 660 (Gly 216-Wat 660 Wat 660-Gly 216 Total of all energies according to type

HN262....017 HNA35.,..0301 H0507....038 HN59,...05014 HN77....OH72 H0143,...OH6’72 H0462...,0165 H0683....0182 HN189..‘.0H675 H0685....0208 O(a)266 HN404 HN404.,..0 (2) 266 0(2)266 HO689 H0689....0(2)266 0(2)267 HO691 H0691....0(2)261 HN279 0(2)308 HN279....0(2)308 NH303....0284 H0476....0301 HN325....0310 H0682....0310 HN320....0353 HN457....0345 H0359....0H699 H0680..‘.0361 H0603....0390 HG604....0421 H0698....0441 H0694. ‘. .OH678 HN379...,0H619 0382 OH619 H0619....0382

MIPHIS Type of interaction D=lr D=4r

MIPHIP D=lr

HB HB HB HB HB HB HB HB HB HB El HB El HB El HB El HB HB HB HB HB HB HB HB HB HB HB HB HB HB VDW HB

1.83 2.52 3.82 1.07 1.12 2.83 1.99 3.96 2.98 3.28 11.15 2.09 11.35 3.66 11.60 3.45 10.29 1.94 1.97 3.44 1.28 1.76 2.13 2.44 3.91 3.82 1.41 0.96 0.87 2.63

El VDW HB TGR

200.68 267.72 79.58 22.74

46.37 271.85 81.00 23.10

250.98 269.41 86.84 23.13

Total

525.24

376.12

584.10

1.83 2.47 4.04 1.07 1.08 3.17 2.00 4.03 2.97 3.31 2.72 2.12 2.77 3.80 2.75 3.87 2.57 2.07 1.97 3.45 1.28 0.98 2.13 2.44 3.97 3.94 1.21 1.01 1.49 1.42

1.83 2.48 4.04 1.17 1.16 3.41 1.98 3.98 2.88 3.17 10.96 2.12 11.36 3.65 11.46 3.82 10.33 1.98 1.97 3.45 1.28 1.90 2.13 2.44 3.80 3.80 1.05 3.86 0.96 3.34

DIPHIP D=4r

D=lr

D=4r

1.83 2.47 3.86 1.17 1.14 3.75 1.99 4.01 2.92 3.14 11.05 2.12 11.39 3.69 11.36 3.95 10.32 1.98 1.97 3.46 1.28 1.69 2.13 2.44 3.91 3.93 1.00 3.88 0.90

1.83 2.49 4.10 1.17 1.13 3.95 1.99 4.02 3.08 3.17 2.69 2.08 2.77 3.82 2.76 4.02 2.57 2.09 1.97 3.48 1.28 1.28 2.13 2.44 3.98 3.77 1.05 3.45 1.62

2.58 -1.15 1.55

1.59

59.12 272.61 84.80 23.10

232.52 272.95 85.62 24.31

54.99 277.50 85.93 25.30

393.44

566.77

393.12

1.83 2.54 4.09 1.17 1.12 3.83 1.99 4.04 3.09 3.24 2.71 2.10 2.76 3.85 2.75 3.95 2.56 2.04 1.97 3.44 1.28 0.88 2.13 2.44 3.92 3.95 0.97 1.78 1.49 1.57

) 1.58

“Energies < 1 kcal mol-’ are not listed for any complex.

Atomic partial charges were assigned to Hip-57 by YET1 according to the AMBER library. The charges on DIPSE were OE (all) - 263 to - 269; PO - 313; P 652; CH (9,13) 139; the others being equal to the ones in Fig. 1. Refinements were carried out with the distance-dependent dielectric parameter set to

166

D= 1.0 r and 0=4.0 r. Different starting conformations with His-57 rotated ? 45 ’ from the position in the refined structure resulted in the same optimized conformation and energy. Table 1 summarizes the energies of interactions between the inhibitor and key active site residues and those between His(His(p)-57) and Asp-102 for each case. Figures 2-4 are stereoscopic views for the refined structures of MIPHIS, MIPHIP, and DIPHIP respectively with a value of D = 1.0 r. As the data in Table 1 suggests, there was perceptible movement of the His(p)-57, Wat-626, and the inhibitor upon changing D to 4.0 r in all three cases. Table 2 gives the energy partition for each refined conformation. Other stabilizing interactions < - 1 kcal mol-’ and the total of each type of energy contribution for each calculation are tabulated in Table 3. DISCUSSION

Electrostatic interactions

The contribution of these interactions differs in the two types of calculations by a factor of 3.97-4.22 as might be expected [ 121. The values at D = 1.0 r, most likely, overemphasize the weight of this component over the other interactions, particularly H-bonding. H-Bonding interactions

These interactions are quite sensitive to the choice of the dielectric parameter where charged species are involved as at the active-site. Shown in Table 1, the H-bond between the primary amine of His-57 and the carboxylate of Asp-102 is insensitive to the structure, and charge distribution of the inhibitor, or the value of D. On the contrary, the H-bond between the H&IA of His-57 and the carboxylate of Asp-102 depends on both the nature of the inhibitor interacting on the other side of His-57 and the value of D. This H-bond is the strongest in MIPHIP due to the interaction of oppositely charged species. The H-bonding energy changes from -2.lto -2.74kcalmol-‘ongoingfromD=l.O r to D ~4.0 r. The values are lower, - 1.73 and - 2.62 kcal mol-’ respectively, for MIPHIS which also reflects a greater response to the choice of D. H-Bonding energies are reduced from - 1.00 to - 2.05 kcal mol-’ when D is changed from 1.0 r to 4.0 r in DIPHIP, which is the most sensitive to the value of D. Inhibitor-Hip-57 H-bonding interaction takes place only in the negatively charged MIPHIP adduct. Although this calculation does not provide for interactions between etheral oxygens and electrophiles, weak interactions at this site are conceivable. The energies of H-bonding interactions between Gly-193 and one electron pair of the PO on the inhibitor are between - 2.3 and - 2.74 kcal mol-l for five of the structures, but only - 1.63 kcal mol-’ for MIPHIP at D= 1.0 r. The slighter H-bonding interaction in the oxyanion hole is be-

167

tween the amine of Ser-195 and the electron pair of the PO of the inhibitor in the range of - 1.76 to - 1.98 kcal mol-l with the exception of MIPHIS that is -0.99 kcal mol-l at D=l.O r. Thus the total energies of H-bonding in the oxyanion hole are - - 4.5 kcal mol-’ except for the case D = 1.0 T in the aged adducts, but with different contribution of the two interactions when His-57 is protonated from the unprotonated. It can be seen in Fig. 4 that the inhibitor in DIPHIP is pulled deeper into the oxyanion hole relative to the other two structures. Some more remote H-bonding interactions involving water molecules are also slighted by the larger emphasis on the electrostatic contribution, but the majority of these does not change at all. Van der Waals’ interactions and geometry Due to the large size of the binding pocket and rotational flexibility of the inhibitor, van der Waals’ interactions were reduced to < 1 kcal mol-’ for most interactions, only a few were 1-4 kcal mol-l and one remained at 15 kcal mol-l. The refinement was not sensitive to a slight change in the geometry of the isopropyl groups in DIPSE. The critical role of the water channel [ 131 leading to the catalytic center is, however, manifest in the motion of Wat-626 and Wat671 at the active-site upon introduction of the second isopropyl group in DIPSE. Energy partitioning As seen in Table 2, the major energy gain on protonation of His-57 in MIPHIP and DIPHIP arises from protein-protein and protein-substrate interactions most of which are located at the active-site. Differences in the contribution of other interactions are minor but very dependent on the value of D. On close scrutiny, it is ostensible that weak interactions are not simply additive in their contribution to the equilibrium state of the adduct. The total energy of stabilization is similar for the adducts with proton on His-57,20-60 kcal mol- ’ lower than for the unprotonated adduct and dependent on the choice of the dielectric parameter. Implications for the stability of serine hydrolase OP adducts In all calculations, the most consistent contribution to the energies of protein-substrate interactions comes from the H-bonding forces in the oxyanion hole in the amount of - -4 kcal mol-l. As might be expected, protonation of His-57 results in an H-bonding energy of again - - 2 kcal mol-l and a sizeable contribution from electrostatic interactions in the negatively charged MIPHIP adduct for which a value of not greater than -5 kcal mol-l would be preferred, as the calculation with D = 4.0 r indicates. Hence, the energies of H-

168

bonding interactions at the active-site for the adduct bearing a positive charge on Hip-57 is N -6 kcal mol-l. This value rises to at least - 11 kcal mol-’ or perhaps larger with the inclusion of electrostatic interactions. Although reliable values for the formation of phosphylated serine hydrolases are not yet available, the equilibrium formation of diethylphosphoryl-AChE from some diethylphosphoryl compounds has been reported to be exothermic by < - 10 kcal mol-l [ 141, about - 10 kcal mol-l more stable than the acyl-AChE intermediate. This strong stabilization is unlikely to stem from only two H-bonds in the oxyanion hole, but is more consistent with stronger interactions such as the ones involved between Hip-57 and an electron rich moiety on the inhibitor. For AChE and the serine proteases, the physical event behind inactivation is likely to be the freezing of His-57 in the wrong protonation state and at the wrong location for general base catalysis of an attack by water at the phosphorus atom. Such restriction would interfere with the proton-switch mechanism. For most phosphyl groups, hydrolysis from the Ser-195 of the proteases is remarkably slow, with half lives in days. It appears that there are only two water molecules close enough at the active-site but they might be blocked from approaching the reaction center. AChE seems to facilitate its dephosphylation somewhat better in most cases [ 151. This ability might be related to a greater closeness of (structural) water molecules for accepting the proton from Hip and for acting in hydrolysis. The phosphylated-serine hydrolase adducts can be analogs of the deacylation TSs if the rate limiting step is after proton removal by His-57 from the attacking water molecule. The second water molecule at the active-site may also assist in tandem in strengthening the nucleophilicity of the incipient water molecule. This mechanism could provide an alternative to the previous propositions [ 161 for the interpretation of multiproton catalysis. A TS for breakdown of the tetrahedral intermediate for deacylation and involving reprotonation of Ser-195 couldalso have some structural analogy to the phosphylated-serine hydrolase adducts, but is less likely to be rate determining. ACKNOWLEDGEMENT

Generous support in meeting the computational needs of this project and in the use of molecular graphics is greatfully acknowledged to Angelo Vedani. This research was supported in part by the U.S. Army Medical Research and Development Command through contract DAMD-17-C-83-3199 and in part by the University of Kansas. REFERENCES 1 2

G.B. Koelle (Ed.), Choline&erase and Anticholinesterase Agents, Handbuch der Experimentellen Pharmacologic, Erganzungswerk XV, Springer-Verlag, Berlin 1963. (a) W.N. Aldridge and E. Reiner, Enzyme Inhibitors as Substrates. Interactions of E&erases with Esters of Organophosphorus and Carbamic Acids, Elsevier, New York, 1972. (b) W.N. Aldridge, Croat. Chem. Acta, 47 (1975) 215.

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5 6 7

8 9 10 11 12 13 14 15 16

W.W. Bachovchin, Biochemistry, 25 (1986) 7751 and references therein. (a) L. PO@, and P. Halasz, Biochem. J., 207 (1982) 1. (b) A. Fersht, Enzyme Structure and Mechanism, andedn., Freeman, New York, 1985, Chap. 1, and pp. 405-413. D.S. Frank and D.A. Usher, J. Am. Chem. Sot., 89 (1967) 6361. I.M. Kovach, M. Larson, and R.L. Schowen, J. Am. Chem. Sot., 108 (1986) 5490. (a) R.M. Stroud, L. Kay, and R.E. Dickerson, J. Mol. Biol., 83 (1974) 185. (b) R.L. Chambers, and R.L. Stroud, Acta Crystallogr., Sect. B, 33 (1977) 1824 and references therein. (c) J. Kraut, Ann. Rev. Biochem., 46 (1977) 331. A. Vedani, J. Comp. Chem., 9 (1988). F. Bernstein, T.F. Koetzle, G.J.B. Williams, E.F. Meyer Jr., M.D. Brice, J.R. Rodgers, 0. Kennard, T. Schimanouchi, and M.J. Tasumi, J. Mol. Biol., 112 (1977) 535. J.W. Pflugrath, M.A. Saper, and F.A. Quiocho, in: S. Hall and T. Ashiaka (Eds.), Methods and Applications in Crystallographic Computing, Clarendon Press, Oxford, 1984, p. 407. SYBYL V-3.2 TRIPOS Associates. J.M. Bianey, P.K. Weiner, A. Dearing, P.A. Kollman, EC. Jorgensen, S.J. Oatley, J.M. Burridge, and C.C.F. Blake, J. Am. Chem. Sot., 104 (1986) 6424. E.F. Meyer, L. Takahashi, and R. Radhakishnan, in: J.J. Stezowski (Ed.), Molecular Structure: Chemical Reactivity and Biological Reactivity, Oxford University Press, 1986, p. 27. (a) J.H. Maglothin and J.B. Wilson, Biochemistry, 13 (1974) 3520. (b) J.H. Maglothin, P. Wins and J.B. Wilson, Biochim. Biophys. Acta, 405 (1975) 370. J.W. Hovanec and C.N. Lieske, Biochemistry, 6 (1972) 1051. R.L. Schowen, in J.F. Liebman and A. Greenberg (Eds.), Principles of Enzyme Activity 9, Molecular Structure and Energetics, VCH, Deerfield Beach, FL, 1987, p. 1.