Structure of a triclinic ternary complex of horse liver alcohol dehydrogenase at 2.9 Å resolution

J. Mol.

Hiol.

19X1) 146, 561-587

Structure of a Triclinic Ternary Complex of Horse Liver Alcohol Dehydrogenase at 2-9 A Resolution H;\Ns EKIX’ND, ,JEAN-J’IERRE SAMAMA~. LEIF WALLI?N, (‘ARL-TVAR BR#NDIZ:N Ikpartmr,,t

of Phe,mistry,

Swedish

S-750

~~niversity

07 Pppsala,

of A4gricultuml

Sciences

Sweden

Ka,rolinska Institute, Holnuvtigen 1 S-104 01 Stockholm 60. Sweden AND

‘I’. ALWYN JONES~ Max-Planck-Institute fiir Biochemir D-8033 Martinsried bei M iinchen ( Grrncan~y (Received

IS July

1980, and in revised form

24 October 1980)

The structure of a triclinic complex between liver alcohol dehydrogenase, reduced coenzyme NADH, and the inhibitor dimethylsulfoxide has been determined to 2.9 A resolution using isomorphous replacement methods. The heavy-atom positions were derived by molecular replacement methods using phase angles derived from a model of the orthorhombic apoenzyme structure previously determined to 2.4 A resolution. A model of the present holoenzyme molecule was built on a Vector General 3400 display system using the RING system of programs. This model gave a crystallographic R-value of 37.9:/,. There are extensive conformational differences between the protein molecules in the two forms. The conformational change involves a rotation of 7.5” of the catalytic domains relative to the coenzyme binding domains. A hinge region for this rotation is defined within a hydrophobic core between two helices. The internal structures of the domains are preserved with the exception of a movement of a small loop in the coenzyme binding domain. A cleft between the domains is closed by this coenzyme-induced conformational change, making the active site less accessible from solution and thus more hydrophobic. The two crystallographically independent subunits are very similar and bind both coenzyme and inhibitor in an identical way within the present limits of error. The coenzyme molecule is bound in an extended conformation with the two ends in hydrophobic crevices on opposite sides of the central pleated sheet of the coenzyme binding domain. There are hydrogen bonds to oxygen atoms of the ribose mojties from Asp223, Lys228 and Hi&l. The pyrophosphate group is in contact, wit,h the side-chains of Arg47 and Arg369. t Present,address: Institut de Chimie, Universitk Louis Pasteur, F-67008 Straxbourg-Cedex, Franre. 1 Present address: Department L’ppsaia. Sweden.

of Chemistry,

Swedish Universit,v

of Agrkultural

Sciences, s-750 07

.Ml

(H)r’l-%R3B/81/0805(i1-“7$lxW/O

(7’ 1981 .Icwlemk

Press Inc. (London)

Lttl.

562

H. FKLVYI) J A

P7’ 2 il,C.

No new residues are brought into the active site compared to the apoenzyme structure. The active site zinc atom is close to t,he hinge region. where the smallest structural changes occur. Small differenres in the co-ordination geometry of the ligands Cys46, His67 and Cys174 are not excluded and may account, for the ordered mechanism. The oxygen atom of the inhibitor dimethylsulfoxide is bound directly to zinc confirming the structural basis for the suggested mechanism of action based on studies of the apoenzyme structure.

1. Introduction Alcohol dehydrogenase from horse liver has been studied extensively with crystallographic methods for a number of years. The structure of apoenzyme crystals has been determined to 24 AA resolution (Eklund et nl.. 1976) and has recently been cr.ystallographically refined to an R-value of 22.5oi, (Jones & Zeppezauer, unpublished work). Furthermore, the binding of a number of coenzyme analogues and inhibitors has been studied in isomorphous complexes of this structure (see BrB;ndBn & Eklund. 1980. for references). These crystals are orthorhombic. space group (‘222,, while ternary complexes with coenzyme, substrate or most inhibitors are monoclinic, space group E’2,, or triclinic (BrB;ndkn, 1965: Zeppezauer et nl., 1967; Eklund, 1976). Additional crystal forms related to these can be found under some conditions (Eklund, 1976), including crystals of alcohol dehydrogenase modified at the lysine side-chains (Plapp et ~1.. 197%). The cell dimensions for all crystal forms are listed in Table 1. In order to study mechanistically important aspects of coenzyme, substrate and inhibitor binding we have now determined the structure of a triclinic complex with NADH and the inhibitor dimethylsulfoxide to 2.9 a resolution. A preliminary account of the low-resolution studies has been published (Eklund B BrGndBn, 1979). This particular complex was chosen mainly because it most reproducibly gave high quality triclinic crystals that diffracted to about 24 14 resolution. We have made an independent structure determination of t’hese crystals using the method of isomorphous replacement,, since we suspected that there might be substantial conformational differences between the apoenzyme and the ternary complexes. The heavy-a,tom positions were derived from difference Fourier maps. using approximate phase angles obtained by molecular replacement methods.

2. Methodology The crystals were prepared in the following way. Two dialysis bags each containing 0.7 to 1 ml of 1% enzyme solution were dialyzed against 10 ml of @OS M-TEST buffer titrated to pH 7.0 with suprapure NH, ; 5 mg of NADH and @fi ml of DM80 were added to each bottle. Distilled 2-methyl-2. 4-pentane diol (MPD, Eastman Kodak) was added during 2 days to a t Abbreviations used: ‘I’ES. N-tris(hydroxymethyl)methyl-Saminoethane sulfonic acid: r.m.s.. rootmean-square; MPI), P-methyl-B,4-pentancdiol: DMSO. ctimethylnulfoxitte; EMTS, ethylmercurythiosalicylste; MIR-phases, Multiple ixomorphous replarement phases: LADHase, liver alcohol plpc~eraldehyde-3.phosphate dehydrogenase: LDHaw. lactate dehydrogenase; GAPDHarte. dehgdrogenase.

ApWTlZpe Ternary cwmplexes Ternary complexes Ternary complexrh Ternary cwnpleses

no 104.4 90 90 no

90 101.9 90 108 100

no 707 72.0 90 !K)

181.7 944 180.7 95 91

50~0 52.0 51.1 56 56i 752 446 44.3 7.5 66

Y( )

f/(X)

x0. of

1 9WOO ” 198.MKI 2 194.0(K) 2 190.()90 1 x1 .ooo

subunits anti vohnes/ asvmmetric unit

BriGden et crl. (19%) Zeppezauer et ccl. (1967) HrBind6n et crl. (19%) Ekluntl (1976) Ekluntl (1976)

Ilef’erenw

564

H. EKLI’NU TABLE

E7’ AL. 2

Compound

Soaking time

(‘onc~n of heavy atom (M)

IJranyl oxalate

I month

10-3

NAl)H

DMSO

2 days Cocrvxtallization Cocrvst~allization

1or3 IV4

NADH NAD+

DMSO EMTS

4 x 1or3

NAD’

4-I -pyrazole

EMTS or Hg VWEMTS 4I-pyrazole

(!omzyme

Inhibitor

The wmcentrations of DMSO and coenzyme in the first 2 derivatives but there was no DMSO in the last 1 derivatives.

were the same as in nat,ive qstals

concentration of 5% and then more slowly until crystals began to appear (7 to 10% MPD). The crystals were then left for 1 week, after which the concentration was increased over a period of 2 weeks to a final MPD concentration of 25%. Preparation conditions for the 4 different heavy-atom derivatives used in the structure determination are summarized in Table 2. Complete sets of 16,344 independent reflexions to 2.9 A resolution were collected for the native complex (enzyme-NADH-DMSO) and for each of the 4 heavy-atom derivatives. All data were measured on a STOE 4-circle diffractometer following essentially the procedures used in the orthorhombic apoenzyme structure determination (Eklund et al., 1976). The data were collected in 4 shells of approximately equal volume in reciprocal space. Several different crystals were used for each shell. (b) d n approximate

model wsing moleedar

rrplacemevd

methods

In the orthorhombic C222, and monoclinic P2, forms, the 2-fold screw axis has a length of 181 A, corresponding to twice the length of the dimer molecule. A primitive cell can be constructed from the orthorhombic cell if the 2-fold rotation axes of the C222, unit cell are neglected. This primitive cell is very similar to the monoclinic P2, cell (Briindbn, 1965). If we remove the screw axis in this monoclinic cell, the c-axis is halved and we obtain cell dimensions very close to the triclinic cell. These relationships were used to approximately position the molecular 2-fold axis in the triclinic unit cell. One of the crystallographic S-fold axes in the orthorhombic form is a molecular e-fold axis, whereas in both the monoclinic and triclinic forms there is 1 dimer in the asymmetric unit. In order to define the molecular axis more accurately, a self-rotation function (Rossmann & Blow, 1962) of the triclinic form was calculated using the 550 largest terms in the resolution range 10 to 49 A. with an integration radius of 30 A. There was only 1 prominent peak in the map of 2-fold rotation. The height was 77% of the origin peak (Fig. 1). The calculation was made with a program kindly provided by M. G. Rossmann. The area around the peak was examined using different rotation angles (x = 170” to 190”) with points sampled in a fine grid to get a more accuratr value of the direction of the symmetry axis. This axis is shown to be strictly 2-fold (X = 180”) with the direction 4 = 30” and 4 = 17”, expressed in polar co-ordinates. These values agree rather well with thosr calculated from thr relation between the orthorhombic and triclinica unit cells. An approximate orientation of the molecule around the local 2.fold axis was obtained by comparing common mercury positions on heavy-atom derivatives of orthorhombic and triclinic crystals. There were 17 well-defined peaks in the triclinir EMTS difference Patterson

HORSE

LIVER

ALCOHOL

DEHYDKOGESASE

Fit:. 1. The %foltl self-rotation function of tric%nic alrohol dehydrogenase represented in polar COordinates. Contours are drawn at levels of 10”” of the height of the origin peak starting at %I”,,. The tlirec%ions of the (8rystalloarsphic b axis and the reciprocal cell axes n* ant1 --(.* are shown.

map. We rotated all these peaks around the local 2-fold axis using the rotation angles given above in order to pick out the peaks that correspond to vectors between 2-fold related atoms. Such peaks should be located in a plane through the Patterson origin, normal to the axis of rotation, since the vectors between related atoms have no component along the rotation axis and therefore are rotated into their centrosymmetric counterparts. Vectors between nonrelated atoms are, in general, rotated into quite different regions of the Patterson spare. Two symmetry related vectors were found which, from their lengths, could be identified with known mercury vectors across the 2-fold axis in the orthorhombic structure. These correspond to the orthorhombic heavy-atom sites C and D where mercury is bound to Cys240 and Cys132, respectively, in each subunit (Eklund et al., 1976). The approximate matrix obtained by this relation was used to transfer the co-ordinates of the orthorhombic model into the triclinic cell with the orthorhombic origin shifted - l/4 in z so that the 2-fold axis passes through the origin. On the basis of this transformation, triclinic structure factors were calculated for the coenzyme binding domain and the catalytic domain separately as well as for 1 complete subunit. The calculation was made for the 4756 reflexions to 45 8, resolution using a factor equal to 7 for all atoms except zinc. t,emperature factor of B = 15 and a scattering Cross-rotation functions (Rossmann & Blow, 1962) between the calculated F-values described above, and the triclinic observed F-values, were then calculated using the same parameters as in the self-rotation calculations. All 3 calculations gave prominent peaks in t,he section x = 190” at (I, and 4 values equal to or close to the values obtained in the self-rotation calculations (Table 3). The same peaks are also present-in the section K = 10 but these peaks are smeared out. The slight differences in these values between the domains suggest possible conformational differences between the structures involving a rotation of the domains relative to each other. We now know that these results are correct and not artifacts of the calculations. The peak for the complete subunit was close to that for the catalyt,ic domain. since this domain is considerably larger than the coenzpme binding domain.

H. EKLUND

E7’AL.

TABLE 3

Results from

rotation function

calculation,s

Self’ rotation Cross rotation : &enzyme binding domain (‘atalgtic* tlomain Subunit Peak positions are given in polar cao-ordinates. (c) Location and rqfinemcnt of heavy-atom parameters Molecular replacement phases derived from a dimer model rotated by x = IgO”, 4 = 17” and 4 = 30” were used to locate the heavy-atom positions. Since we were uncertain at this stage about the validity of the conclusions concerning conformational changes, we assumed no conformational differences between apoenzyme and ternary complex in these calculations. The rotated apoenzyme model had an R-factor of 55% for all reflexions to 4.5 a resolution. Calculated phases were used to compute difference Fourier maps to 4.5 A resolution of the uranyl, Hg(CN)42- and EMTS derivatives. The highest peaks in each map could easily be related to vectors in the corresponding difference Patterson maps. The agreement obtained was excellent and all the sites were located for all derivatives, as was later checked by difference Fourier maps using MIR-phases. By this procedure the different derivatives were also correlated to the same origin. The heavy-atom positions in the 4-iodopyrazole derivative, which was not used in the 4.5 A resolution study (Eklund & BriindBn, 1979) were deduced from difference Fourier maps using isomorphous phase angles from refined positions of the other 3 derivatives. The heavy-atom parameters were refined using a program originally written by M. G. Rossmann (Adams et al., 1969). The phase analysis was carried out by the method of Blow & Crick (1959) with alternating cycles of phase calculations and least-squares refinement (Dickerson et al., 1961). During refinement the “best” phases were used, since the phase information from all derivatives were then better balanced (Jack et al., 1976). This method gives lower occupancy values for the heavy atoms and, as a consequence, higher values of R-modulus. After 10 cycles of refinement of the 4.5 A data an electron density map was computed that contained large peaks at some of the heavy-atom sites. These could not be removed by further refinement. In order to analyze possible systematic errors in the data, we computed scale factors between native and derivative data for groups of reflexions as a function of h, k, I, increasing resolution and increasing magnitude of the F-values for each crystal. For some of the derivative crystals we observed a large systematic trend in these values as a function of resolution which, in addition, was clearly anisotropic. Correction factors to eliminate these systematic trends were obtained by minimizing the quantity E = 2 (Fp- F;,)‘, where F HP= S x FHP x exp - (h’u, + k2u2 + t2u3 + klu, + hlu, + hku,), S is the scale factor, U, to ug are the anisotropic correction parameters, F, is the observed native F-value and FHP i s the observed derivative F-value. For some of the derivative crystals the u-values were considerably larger than the standard deviations. Further

HORSE

LIVER

ALCOHOL

DEHYDKOGENASE

.567

refinement of the heavy-atom parameters using the corrected data produced an electron density map with no significant disturbances at the heavy-atom sites. Anisotropic correction factors were subsequently applied to all derivative crystals used at higher resolution. One possible cause for the need of these correction factors might be that the heavy-atom binding causes anisotropic disorder in the crystals. Alternatively, they may correct for systematic. errors in the data collection. The addition of a 4th derivative (4.iodo-pyrazole) gave significantly better phases for a numbrr of reflexions. and also a better balance between the derivatives. The mean figuna of merit) increased by about til. Refinement at higher resolution was carried out by dividing the rrfexions in shells of increasing resolution at 3.7. 3.2 and 2.9 A. These shells of data were independently refined, using as starting parameters the final values obtained from the refinement at the preceding lower resolution. All data within each shell were included in thr rrxfinement. which was continued until all shifts were about half t,he standard deviations. Some refinrment results are given in Tables 4 ancl 6. (d) Low resolution

structwel

L\n electron density map at 4.5 A resolution based on 3 derivatives was first calculated in sections perpendicular to the non-crystallographic 2-fold molecular axis. An orthorhomhic* isomorphous map was calculated at the same resolution and in the same orientation. In the orthorhombic map, the n-carbon atoms were plotted and connected to show the main-chain tracing. The triclinic map was interpreted by comparison with the orthorhombk model as

Fkal

h,cn!vy-atom

parameters

of triclinic

Site?

.*

A I-3 A’ B’

0+3134 0.55213 Cl.5459 w5t-349

k‘ k” I) I)’ E L.I P F

@7450 05939 0.1762 o%okiki @1686 0.0336 0.1305 ml231

( (” I) I)’

@7408 05920 @1785 OWO7

E b.I

0.1742 044%

liver

alcohsol dehydrogrn~ae

Cnel heavy-at,om paramet,ers and occupancies in the 3 shells used for refinement of &a from 4% to 29 A resolution. The refinement results of the low-resolution data have been reported earlier (Eklund & HriindBn. 1979). t The sites with the same letter are symmetry-related by the non-c~r~stalloyrapllil Z-fold axis. P and P’ are DMSO sites with negative oc~vupancy.

568

Figure of merit Number of reflexions r.m.s. Et I’ranyl r.m.s. f W,,) EMTS r.m.8. E cocar,vstallized r.ni.s. f ff (“0) EM’I’S r.rn.s. E soaketl r.m.s. f k (“0) 4~I-pyrazole r.m.s. E r.m.s. f K (“,,) t The terms r.m.s. E. r.m.s. f and K are definetl by Siitlerberg et nl. (1974) described earlier (Eklund &, BrB;ndBn, 1979). The relation bet,ween the orthorhombic coordinate system and the triclinir map was obtained by superposition of the coenzyme binding domains. Approximate a-carbon co-ordinates for the catalytic domain were then measured from the triclinic map. These were compared to the corresponding orthorhombic 2carbon model co-ordinates with a least-squares program (Rao Rr Rossmann, 1973). Both catalytic domains of the triclinic structure required a rigid body rotation of 75” for superposition. The mean deviation between the r-carbon at)oms in the super-imposed catalytic domains of the 2 structures was 1.8 A and the corresponding r.m.s. value was 2.1 A. These differences reflect to a large extent the errors in the approximate co-ordinates of the triclinic structure at that stage. (e) High-rasotution

modPI building

An MIR electron density map was calculated at 2.9 a resolution using 14,342 reflexions. The model building was carried out on a Vector General 3400 display system using the RING system of programs (Jones, 1978). The molecular envelope of each subunit was blockcontoured at levels of I and 3 standard deviations above the mean value. The orthorhombic mode1 of the dimer was modified by rotation of the catalytic domains as described above and positioned in the triclinic unit cell. Except for the regions between residues 294 and 297, this starting model was within 2 a of its density. Such shifts are easily made using the model building program FRODO which, among other things, allows one to distort and modify a model by breaking any bonding connections and then shifting and/or rotating a group of connected atoms into density. Good stereochemistry is maintained by immediately regularizing the model. while defining that certain atoms remain fixed in density. The quality of the map was good. The main chain was generally easy to follow. but contained breaks at some points, especially in helices at the surface and at glycine residues. There were, however. no doubts about the chain tracing at any point. The general character of the side-chains was usually obvious. Most aromatic side-chains had large flat electron densities. Almost all sulphur atoms were in regions of the higher contour level. Valine, threonine and leucine residues usually had a suggested fork shape. There was defined electron density for all parts of the coenzyme molecules but the density at this resolution

HORSE

LIVER

ALCOHOL

DEHYDKOGENASE

MO

allowed some flexibility of the model. The 4 zinc atoms of the molecule and the phosphate groups of the coenzyme were at the highest peaks on the map. The inhibitor DMSO was clearly visible in both subunits with high electron density. Structure factors were calculated from the final model of the protein, coenzyme and zinc* atoms using the PROTEIN program of Steigemann. The DMSO molecules were not included. A crystallographic H-factor of 37.9% was obtained for the 13,018 reflexions in the range 5.0 to 2.9 A using an overall temperature factor of 17. Only atoms in density, corresponding to XW,, of the t)otal number of atoms in the molecule, were used for this calculation,

3. Description of the High Resolution Model The gross structure of this ternary complex molecule is thus rather similar to t)he apoenzyme structure, Each subunit is divided into a coenzyme binding domain and a catalytic domain. The coenzyme binding domains of each subunit are joined together into a central compact core of the dimeric molecule. The catalytic domains are at the ends of the elongated molecule, which has approximate dimensions of 10 .A x 55 w x 100 ‘4. The active site regions are in the clefts between the central core and the catalytic domains. A schematic diagram of the polypeptide fold and the bound zinc atoms is shown in Figure 2. For detailed description of the apoenzyme structure and the nomenclature used to describe the fold of the polypeptide chain, see Eklund et al. (1976). Here we will of the conformational differences between the present (Loncentrate on a description structure and the apoenzyme structure as well as on coenzyme and inhibitor binding. We will also discuss some implications of the new structural knowledge on the mechanism of action of liver alcohol dehydrogenase.

FIN:. 2. A schematic drawing of t,he dimeric LADHase molecule. One subunit is shaded. The central part of the molecule is the 2 coenzyme binding domains bound together across a &fold axis perpendicular to the plane of the paper. The 1 catalytic domains are separated from this central part b) 2 clefts. (IVe are indebted to Bo Furugren for the drawing.)

570

H. EKLUNII (a) Conformational

F?' 1 AL.

difference.q between apoenzyme

and ternary

complex

We wanted to obtain an initial objective measure of the conformational differences in the dimeric molecule, which should be independent of the particular regions that are aligned. A comparison of vectors between pairs of chemically identical atoms in the apoenzyme and ternary complex should give such a measure. Figure 3 shows a plot of the differences between the lengths of such vectors as a function of residue numbers. It is apparent from this plot that the relative orientations between atoms in the coenzyme binding domains are very similar in the two structures, whereas many atoms in the second domain differ. It is also obvious that residues 294 to 296 in the coenzyme binding domain are subject to a local conformational change. The two coenzyme binding domains have thus preserved their relative orientation, whereas the two catalytic domains have rotated relative to each other. In order to analyze these differences in more detail we have superimposed corresponding n-carbon atoms in the coenzyme binding domains of the two structures by a least-squares rigid body rotation (Rao & Rossmann, 1973). The matrix obtained was then applied to all atoms in these structures and the positional differences of corresponding atoms analyzed. Figure 4 shows a plot of these differences for the r-carbon atoms as a function of residue numbers. Table 6 gives some statistics of these comparisons for all atoms in defined density. In order to analyze possible internal conformational differences within each domain, we compared the positions of corresponding ‘t-carbon atoms after superpositioning of individual domains. These results are given in Table 6 and Figure 5. The main conformational differences between these t’wo forms of alcohol dehydrogenase can be described as a rigid body rotation of each catalytic domain by 7.5” with respect to the central core of the dimer. Coupled to this rotation is a local movement of the loop region comprising residues 294 to 297 in the coenzyme binding domain. These conformational changes could be detected already at low resolution (Eklund & Branden, 1979) and are confirmed and detailed by the present higher resolution study (Fig. 6). It is striking how similar the internal structures of the catalytic domains are in the orthorhombic and triclinic forms (Fig. 5). The conformational change is thus almost entirely a rigid body rotation similar to what has been found for immunoglobulins (Huber et al., 1976) and hexokinase (Bennett & Steitz, 1978). An inspection of Figure 4 shows that the regions of minimum change in the orientation of the catalytic domain relative to the coenzymr binding domain are around residues 44 to 46, 67-68, 93 and 110. where the differences are only of the order of 1 A. These residues are centered in one region of the molecule and thus define a hinge region for the rotation. This hinge region is essentially the hydrophobic core K4 between helixes ~2 and ~3. It, is significant’ that these helices also form the covalent connections between the two domains. A structurally similar hinge region has been observed in tomato bushy stunt virus (Harrison et al.. 1978). Seven hydrophobic side-chains from helix 13, six from helix r2 and. in addition, three from the pleated sheet region /31 are packed in this core. The rotation of the catalytic domain can be accomplished by a small sliding movement of these two

I

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I

I

I

I

3

<, -

I

I

I

I

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--R-

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-8

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--9

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--cP

-R /

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_-

;,i:i

HORSE LIVER ALCOHOL DEHYDROGENASE TABLE~ IJiffwrrnws

bctwern PI positions after superimposing srparatr subunits of the two strwctwes

Subunit (‘w VW (‘at (‘at

I)ifftwncrs

I)ornnin (he (‘at C’lW (‘oe (‘oe* (‘cw* (*at (‘at

1

‘I’1 ‘I’.’ Ti ‘I’.’

0 0 0 0

in the positions

Subunit

Subunit 2

1

Average difference (A)

r.m.s. t1ifferenc.e 0)

0.77 0~70 @64 04%

Subunit 2

Number 01’ atoms ~mparrtl

0.08 WI3 0.71 OS.5

of all atoms in, separate domains coenzyme binding domaius Average difference (a)

domTaitrx from diffurrtt

14-l 114 “31 231

after superimposing

thr

r.m.5. tlifferenve (.S)

‘I’1 1’1 ‘I’1 ‘I’:! ‘1’1 T” ‘I’1 ‘1’2

(‘oe. coenzyme binding domain ; (‘at, catalyt,ic domain: Tl. triclinic chain 1 : T%. tridink orthorhombic, chain: Cue*, Cue where residues 293 t,o 298 have been deleted.

X70 13x4 x40 x54 807 t-k!5 1480 1355 chain 2 : ().

heiices without any large structural changes within the domain. An exception is the interaction area between the two domains furthest away from the hinge region where the largest movements occur. Here residues 294 to 297 of the coenzyme binding domain must be moved away in order to make room for the new positions of residues 51 to 58 of the catalytic domain. Stereo views of this region in t)he two struct’ures are given in Figure 7. The n-carbon atom of Pro296 is changed 5 a in both subunits and the y-carbon of this residue as much as 8 to 9 a. A remarkable feature of the conformational change of this loop is that, the side-chain of Val294 point,s in opposite directions in the two structures. In the apoenzyme structure it point,s away from the active site cleft, whereas in the ternary complex it is directed into t,his cleft and is in contact with the nicotinamidtx ribose. As a result of these movements, the interaction areas bet’ween the domains have changed considerably. In t,he apoenzyme structure there is only weak contact) betwren a few residues from each domain in t,his region (see Fig. 7). Residues 55-3.5 are in contact with residues 296-297. In the ternary complex these parts havta moved so that the contact area is shifted by about 5 A, and involve more residues in t’hr same region (Fig. 7). The closest contact is a hydrogen bond between the mainchain carbonyl oxygen of Thr56 and t,he nitrogen atom of Asp297.

I

I

.

20

60 80 100

120 140

FIG. 5. Distances between equivalent CI atoms of the catalytic after these domains had been separately superimposed.

40

320 domains of the ape and holo forms

160

. , . . . . . . . .

340

I

360

HORSE

LIVER

ALCOHOL

I)EHYl)I
(b)

FIG. 6. Stereo diagram of the subunit of (a) apoenzyme (thick lines). (b) ternary wmples (thin lines) and (t,) thr :! strwtures superimpowtl. The Ca atoms of the wenzwnr binding domains were used for the qwrlwsition.

A striking example of the drastic change in this interface region het,ween the domains is that the side-chains of Leu57 and \‘a1294 are about 15 a away from each other in the apoenzyme but only about 4% A in the ternary complex. From Figure 4 it is seen that there are also large structural differences at the surfaces of’ t)he catalytic domain. with a difference of 7 w for Arg133. There are several sidrchains at the surface which differ by more than 10 .A.

H. EKLITND

E’?’ AL

(a)

(b)

FIG. 7. Stereo diagram of’ the (*left region between t,he 1 domains in (a) the apoenzvme. (b) the ternary complex and (c) both structures superimposed. The ternary complex is in red.

(b) Aubunit

The main hydrophobic of equivalent side and 312 area are very

subunit interaction area in the centre of strands of extended to 317 on the other similar in the two

interaction

in the enzyme dimer consists of a large the molecule surrounded by two antiparallel pairs chain of each subunit ; residues 298 to 304 on one side (Eklund et al., 1976). The interactions in this structures.

HORSE LIVER ALCOHOL DEHYDKOGENASE ((2) 1)ifferences

between

the two sttbunits

577

of th,e molec~rIr

The two chemically identical subunits of the molecule are related by a crystallographic 2-fold axis in the orthorhombic crystal form and thus are structurally identical. In the triclinic form, however, the whole dimeric molecule is in the asvmmetric unit and the two subunits need not’ be in the same conformation. The two’subunits were therefore examined for possible structural differences. Aft,er superposition. the average difference for the 374 n-carbon atoms was found t,o be (b;i a and the r.m.s. difference 0+7-4. The average difference for all defined mainchain and side-chain atoms is 0.7 A and the r.m.s. difference is 1.0 .h between the two subunits (Table 6). These calculations show that t’he t’wo subunits are \-er:\ similar. A close inspection of differences larger than 3 A shows, as expected. that, these are mainly found for long charged side-chains at, the surface.

(d) (‘oenryme

binding

The enzyme binds one coenzyme molecule, SADH. per subunit and the occupancies at the two sites are very similar as judged from the height of the map density. Within the limits of errors of the present model, the conformations of the two coenzyme molecules as well as their interactions with the protein subunits are similar. Interactions between coenzyme and protein are listed in Table 7. and some of these are illustrated in Figure 8. The adenine ring is sandwiched between the side-chains of isoleucine residues 224 and 269 (Fig. X(a)). The ring is partly shielded from solution by the side-chain of Arg271 in both subunits. N-10 of adenine is within hydrogen bond distance of the side-chains of Arg271 and Asp273. The adenine occupies only a small part of the surprisingly large cleft where it binds. ()ne of the few structurally equivalent residues that are also chemically identical in the coenzyme binding domains of the known dehydrogenasrs (Rossmann et al.. 1975) is an aspart,ic acid (Asp223 in LADHase). This residue hydrogen bonds to O2’ of the adenosine ribose. The ribose has been built in C-2’.endo conformation which both fits the density and avoids too short contacts between O-2’ and Asp223. The second hydroxyl group of the ribose, O-3’, is at hydrogen bond distance to the amino group of Lgs228, which is in well-defined densit,y. (‘hemical modifications (Plapp et ~1.. 1973: Dworschak et ul.. 1975) of this residue increase the turnover number for alcohol oxidation tenfold due to an increase of the dissociation rate of t*he enzyme-coenzyme complex. These modified enzyme molecules have been orvst~allized (Plapp et al., 1978a) and are now being studied by X-ray methods in order to obtain a molecular explanation for this rate increase. The phosphate group of the AMP part of the coenzyme is close to t)hr quanidinium group of Arg47. The second phosphate is within possible contact with t’he guanidinium group of Arg369 at a somewhat longer distance. This arginine is positioned bet,ween the phosphate and the side-chain of Glu68. The salt-bridge between Arg369 and the internal residue Mu68 that is present in the apoenz\-mt> structure (Eklund et al., 1976) is still present in this structure. The oxygen atoms of the phospha’te are also at possible hydrogen bond distances from the nitrogen

578

H. EKLITND TABLE

Enzyme-rornzymP

(‘oenzyme moiety Adenine

interactions

LADHasr Phe198 \‘a1222 Asp223 YIle2*‘4 HeOtiS

E7’ AL.

7

irl 1Al)Hase.

Residues LDHase

LDHnsr

GAPDHaxe

‘I’?‘pe of cY,ntact

Va127 \‘a152 Asp53 \‘a64 Tyr85 IleHti Ala98 Ilel19 Ilcl2:~

Asnti Ad1 Asp32 Phd4

Interior of pocket Interior of pwket H-bond to ring N Hydrophobic. H-bond to ring N Hydrophobic, Hydrophobic, Hydrophobic, Hydrophobic H-bond to N-G

Gly28

Gly7 Asp3& (‘ = o-x

Thr9ti Phc=99

Arg’7 1 Adenosinc ri hose

Glyl99 AspZ13 Lys228 IldOo

ZB Lys58 Asp30 rvlet~5.i

LeulXi Pro 1xx S-10 s-11 AsnlXO

Pyrophosphate

Nicotinamide ribosr

(’ = O-269 N-294 His51 Gly270 \‘a1203 Glv293 rallS1

Niwtinamide

va1203 \‘a1292 \‘a1294 Thr178

(‘arboxamitlr N-319

rind (:=I I’DHasu

(‘ = O-98 N-140 Glv!)!) mz Thr97 Pro139 \‘a132 \:a1138 Asnl40 Leu 107 ThrMi Ile2.50 (’ = Cbl39 Srrlti3

I‘nderlined residues on the same line are structurally Rossmann rl nl. (1976).

Gly99 Ah31 (’ = O-96 Anion Serl19 b(:lvoi

H-bond to 0-2’ H-bond to O-3’ H-bond to O-3’ Hydrophobic Hydrophobic, from djawnt subunit H-bond H-bond Charged (‘hargetl

to main chain to main chain or polar or polar

H-bond to O-3’ H-bond to O-L” H-bond t,o O-3’ Hydrophobic

Ala120

Hytlrophobi~~ Hydrophobic,

Ilell

Hydrophobic

‘I’,vr317

( ‘ys I 4!3

(‘hargr transfer

Asn313

H-bond H-bond

equivalent

awonling

to the alignment

I,?

HORSE

LIVER

ALCOHOL

--

.

(b)

DEHYDtiOGENASE

579

580

H. EKLlTND

E7’ AL

(dl

PIG. 8. Diagrams illustrating interactions between NADH and electron density sandwiched between the side-chains of Ile224 densities. (b) Interactions between protein side-rhains and Surrounding of the nicotinamide part of NAD. (d) Stereo diagram coenzyme (in red).

LADHaue. (a) The adenine ring in its and Ile269 in parts of their electron the ADP-ribose part of NAD. (v) of Cn atoms of one subunit and bound

atoms of Gly202 and Va1203. O-3’ of the nicotinamide ribose is within hydrogen bond distance of the side-chain of His51 and O-2’ hydrogen bonds to the main-chain nitrogen atom of residue 294. The nicotinamide moiety is hydrogen-bonded through the amide group to the nitrogen atom of residue 319. The ring is at van der Waals’ contact with Thr178 but the carboxamide is not in hydrogen bond contact to this side-chain as suggested from the low-resolut,ion structure (Eklund & Brand&r, 1979). No atom in the coenzyme molecule is closer than 4.5 A from the active site zinc atom. The structure of the coenzyme binding domains of the dehydrogenases LDHase, GAPDHase and LADHase are very similar, and could be superimposed by aligning equivalent residues (Rossmann et al., 1975; Ohlsson et al.. 1974). By transforming the available co-ordinate sets (Brookhaven Data Bank) of LDHase and GAPDHase to the orientation of one subunit of LADHase using n-carbon atoms of the NADbinding domains, the binding mode of NADH in LADHase could be compared in more detail to that of LDHase and GAPDHase. As seen from Figure 9, the general

HORSE

LIVER

ALCOHOL

DEHYDKOGENASE

(a)

(b) WI<:. 9. Stereo diagrams showing the wnf’ormation of’ coenzyme bound to LADHase (thi(,k lines) compared to (a) LDHase and (b) GAPDHase (thin lines). Structurally equivalent CT atoms of’ the NAI)binding domains have been used to obtain the superposition of’ the coenzyme m&c-ules.

conformations and positions relative to the coenzyme binding domains are very similar. The existing differences depend mainly on differences in the size of the sidechains that line the coenzyme binding sites. The nicotinamide conformation in GAPDHase is of course different. The specific interactions also show a number of similar features as seen in Table 7. One intriguing (Ho1 et al., 1978) similarity is that the pyrophosphate moiety in all three structures is attached by hydrogen bonds to main-chain amino groups of structurally equivalent residues at the amino end of helix %B (Fig. 10).

582

H. EKLUND

E7’ AL.

FIG. 10. Picture from the display system illustrating the interaction between the pyrophosphate moiety of NADH and helix aB of the roenzyme binding domain of LADHase.

(e) Dimethylsulfoxide

binding

The inhibitor DMSO binds with similar occupancy in the two subunits. The oxygen atoms of the inhibitor bind directly to the zinc atoms, thereby forming a roughly tetrahedral co-ordination around zinc together with the three protein ligands Cys46, Cys174 and His67 (Fig. 11). One of the methyl groups is in contact with the nicotinamide ring. Short contacts from the protein and the coenzymes to the inhibitor molecules are listed in Table 8. The distance from C-4 of the nicotinamide ring to the sulphur atom of DMSO is around 5 A.

4. Implications

for the Mechanism of Action

The conformational changes observed here have rather small effects on the structural arrangements of the protein atoms that are at, the active site (Table 9). The ligands to the active-site zinc atom, Cys46, His67 and Cys174 as well as some important residues in the substrate binding pocket, Phe93 and PhellO, are close to the hinge region where the structural changes are less than 1 A. Thus no new residue has been brought into the active site and no change of ligands to the active site zinc atom has occurred. The structural model for coenzyme and substrate binding that was deduced on the basis of inhibitor binding to the apoenzyme structure (Eklund et al., 1974; BrB;ndBn et al.. 1975) has thus been shown to be essentially correct by the present study. This confirms the structural aspects of the suggested mechanism of action (Eklund et uZ., 1974; Briinden et aZ., 1975) for this enzyme iYhich was based on that model. The obvious question to ask is then: What is the effect of the conformational transition described here on the catalytic mechanism ! At the present stage of the structural work we can observe the following effects. In the apoenzyme structure the active site zinc atom is accessible to solution from two different directions. One of these is through the substrate binding pocket (Eklund et aZ., 1976). Perpendicular to this direction is the cleft region where the NMN-part of the coenzyme binds and where the largest conformational changes

HORSE

LIVER

ALCOHOL

I)EHYI~IIOGGNASlC

(a)

J

FIG. II. St,ereo view of the substrate binding pocket of LADHase. (a) The apoenzyme. (b) The ternar>wmplns. (c) The apoenzyme (in black) and t,he ternary cwnplex (in red) superimposed.

584

E’f’ AL

H. EKLITND

TABLE X Intrmctions

involving

the

irrhihitor

molecrrlr

dimethylsrrlphoridr

Atom in DMSO

Protein or coenzyme atom

0

Zfl SG 46 (‘I3 48 (‘I):! (ii NE2 07 CEI 67 SC 174

s

zn

IGstance (A)

SE2 Ci7 Cl32 93

(‘71 9s .* (‘I)1 141 (‘4N NAI)H (‘1

(‘I3 4x (“I)1 141

(‘2

(‘3N (‘7N SlN 01N

NADH SADH NADH NADH

TABLE 9 !#w

largest

residues

lining

Residue Ser48 Leu57 \‘a158 Phe93 Phello Leul16 SW1 17 Leu141 Pro296 Ile318 Met30ti Leu309 tier310

differmces between chemically equivalent the substrate binding cleft in the aye md holo forms of LA IlHasp

structural

(“(1 subunit 1

C% subunit 2

Side-chain subunit 1

2.3 3.0 3.5 1t5 0.7 29 2.1 3.4 5.0 1.1 0.5 0.4 0.1

2.3 3.0 3.7 2.1

2.3 5.1 3.3 2.3 13 5-3 4.5 4.5 8.4 2.0 I .7 3.4 0.3

0% 1.4

2.1 2.7 5.2 0.9 0% 0.5 0.4

Side-chain

subunit 2

HORSE

LIVEI<

AI,(‘OHOL

I)EHYI)tZOGENASE

5ri:i

wwr. This cleft, is completely closed off as a combined result of coenzyme binding and the conformat,ional change of residues 17 to 57 and 294 to 297. The active sit’e is thus considerably more shielded from the solution and in a more hydrophobic environment, in this holoenzyme structure than in the apoenzyme structure. An incwase of the hydrophobicity around the positively charged nicot,inamide group in the ternary enzyme -SA41)+~alcohol complex would faciiitat,e hydride transfer from zillc-hound alcohol to X,41)+. analogous t,o the oil-watjer--hi&dine mechanism suggested for LDHasc 1)~ Parker & Holhrook (3977). Th’ IS suggestion is reinforwtl 1)~.the fact that structurally homologous regions in space in relation to the aetivcb sit,e arc warred in both LDHase and LAI)Hase hy the very different (,otlformational changes (Gklund & BrGndb, 1979). l,igand-induc~c,cl wnformat,ional changes of enzyme molecules have Iws suggested 1)~ Koshland (1959) to play an important role in enzyme catalysis. The large strwtural difference observed here between the ape and holo forms of 1978). I,;\ DHase is indwed 1)~ coenzyme binding (BrGndbn & Eklund. Tl~ern~odynami~ studies reported by Sulwamanian & Ross (197X) have sho\vn a large entropic~ effect, upon binding of coenzyme t’o L.AT)Hase in contrast to LI)Hasr ~vhere t’he essential conformational differencr involves only a small flexible 1001) region (White vt (~1.. 1976). The molecular basis of t,he ordered mechanism (Theorell & (‘hawe. 1951 ) has been impossible to dednce from the structure of the apoenz~mv (13r6nd6n & ICklund, 1!)78). Binding of subrate molecules is mnch stronger to the ~*omplexrs wit,h wenzyme t’han to apoenzyme. in spite of the fact that) there is no steric tlindranc~e for substrate binding in the apoenzyme structure (Brhldbn rf trl.. I!)75). Recent spectroscopic studies (Maret it 01.. 197!): I)ietrich P/ nl.. 1!)7!)) in cwmbinatjion wit)h the present X-ray studies have provided a first approximation to an understanding of this mechanism. Maret rt rrl. (1!)79) haye selwtivel~ sllt)stit,ut,ed the catalytic zinc atom by ot’her metals such as whalt. cadmium and cvq)lwr. The same group have also shown t’hat) there are drastic changes in thv vc)l)alt spectrum when the binary complex with roenzyme is formed ( 1)ictrich PI rrl. 1079). The present X-ray studies have shown that the coenzyme molec~ult~does Ilot Ijind to zinc. and that the same ligands to zinc are present in the two structures. Tt~tw sl)t.(,trosc.ol)ic. studies t)hus strongly indicat,c that changes occur ~II t,tw CT)ordination geometry whic4r affect the electronic properties of tjhc act,ivr site metal atom. 1’resumal)ly. the cot~nz!-me-ind~rced c~onforrnational change drscrilwd hew tnodutatjes the properties of the active site zinc atom so that it is more al+ to COordinatr and activate the substrate. Detailed studies to high resolut,ion of’ the Ibinding of different substrates are now underway and the results wilt 1~ discrrsstvl it) a sulwt~qiwnt palwr. The sul)stjrate binding pocket of LXI)Hase (Pig. 1) is lined with a nurnI)t*r of’ hydrol~hohi~ residues. The lwsit,ions of these side-chains are affect,vd to varying ctegrec~sI)y the conformational change. as seen from Figure 1I and Table 9. The changes in the t~opology of the pocket might be important for a l)recise tit of larp~ sul)stratrs. \vhich would reflect the l~h~ysiological function of this enzyme. On t,he basis of distances calculated from nuclear magnetic IIW)I~~I~W rneasrlrements of c:ol)alt-substitutetl LADHase, it, has been suggest,ed (Sloan it (I/.. 1!)75) that, the oxygen atom of the substrate binds indirectly to zinc vitr a zilic*‘?3

5%

H. EKLL’XD

61’ .-lb

bound water molecule. These experimental measurements hare been repeated and confirmed 1)s Andersson et a,l. (1979) using the specifically cobalt-sul)stituted LADHase by Dietrich et ul. (1979). b’mthermore. Dr.vsdale & Hollis (personal communication) have obtained similar results for complexes \\ith NADH and DMSO as well as with SAD+ and trifluoroethanol. ln our X-ray studies we observe the following situation. The electron density obtained for the DMSO moleculr at 29 4 resolution unambiguously shows that this molecule is directly bound to zinc. \c’e have also pre\-iously shown at 2.9 A resolution that) imidazole binds directly to zinc in the apoenzyme st,ructure (Hoiwe ct Rr&ndCn, 1977). Furthermore, we hare found from a low-resolution study of ternary caomplexes with hromobenz~lalcohol and trifluorethanol (Piapp et al., 19786) that these maps can be intrrpreted only in terms of direct binding of substrate t,o zinc. A high-resolubion map of the bromohenzyl alcohol complex confirms this interpretation (Eklund et /I/.. unpublished observations). There is clearly a discrepancy between the interpretations of the X-ray data and of the nuclear magnetic resonance results. It is possible that the experimental data hare not been interpreted correctly in one case or the other. The approach used by resonance data Sloan rt al. (1975) to obtain distances from the nuclear magnetic relies heavily upon small differences between relaxation rates and has t,een subject of the ?i-ray maps are. to severe criticism (Burton et (I/.. 1979). Th e interpretations on the other hand, unambiguous with regard to distinguishing between direct or indirect, binding of the ligands to zinc. The two cases require a posit~ional diffGrenrc of about 2.5 .A between the cent,res of t,he bound molecules. and this difference can he discerned readily at the resolution obtained. Since the enzyme in the t’rictinit crystals is catalytically active (Bignetti et rrl.. 1979). it’ can be assumed that t’hc st,ructures of the enzyme complexes are the sa,mc in the crystals and in solution. 1Vr wish to express our gratitude to Professor Robert Huber for t,he display facilit,irs placed at our disposal. We are grateful for the skilful technical assist,ancr of Jutt)a Schubert and Gunilla Lundquist. This work was supported by grants from thr Swedish National Scirnc~e Research Council (grant no. 2767) and to one of us (J-l’. S.) from thr (‘entre National de la Rrcherche Scient,ifique.

REFEREVVES I Adams. M. J., Haas. D. ,J., Jeffery, B. A.. McPherson. A.. Jr, Mrrmall, H. L.. Rossmann. $1. (i., Schevitz. R. W. & Wonacott, A. J. (1969). J. divot. Rio/. 41, 159-188. Andersson. I., Burton, D. R.. Dietrich. H.. Marrt, W. X: Zrppezauer, ,&I. (1979). In Metalloproteinn (Weser.. U.. ed.). pp. 246-253. Georg Thieme Verlag, Stuttgart and New York. Bennett, W. 8.. Jr & Steitz. T. A. (1978). Proc. Sat. .-lead. Sci., I’.S..-I. 75. 1848-18%. Bignetti, E., Rossi, G. I,. & Zeppezauer, E. (1979). FEES Letters. 100. 17-22. Blow. D. M. d Crick, F. H. C. (1959). Acta Prystallogr. 12, 794-802. Boise. !I’. N: BrB;ndb, C.-I. (1977). Ectr. J. Riochrm. 77. 173-179. BrGndGn. (‘.-I. (1965). .-lrch. Riochem. Riophys. 112. 215-217. BriindGn, C.-I., Jiirnvall, H.,. Eklund, H. & Furugrrn, B. (1975). In 7Thr E’nzymen (Boyer. I’. D., rd.), vol. 1 lA, pp. 103-190, Academic Press, New York. BriindBn, C.-I. & Eklund, H. (1978). In Molecular Interactions and Activity in Proteins, Ciba Foundation Symposium 60 (new series), pp. 63-80, Excerpta Medica. Amsterdam.

HORSE

LIVER

ALCOHOL

DEHYDI~OGESASE

5x7

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Huber. R.. Deisenhofer. J., Colman, P. M.. Matsushima. M. Hr Palm. W. (1976). AYrct/(re (Imdo7r), 264, 41.5420. ,Jark, A., Ladner, ,J. E. & Klug, A. (1976). J. Mol. Riol. 108, 619--649. ,Jones, ‘I’. A. (1978). J. .4ppZ. Crystallogr. 11, 268-272. Koshland. D. E. (1959). In The Enzymes (Boyer, P. D., Lardy. H. & Myrback, K., eds), 2nd edit. vol. 1, pp. 305-346, Academic Press, New York. Maret, W., Andersson. I., Dietrich, H., Schneider-Bernliihr. H . Einarsson, R. & Zeppezawr. M. (1979). Eur. J. Biochem. 98, 501-512. Ohlsson, I.. Nordstriim, B. & BrLnd&n, C.-I. (1974). J. Mol. Rio/. 89, 339~-354. Parker. D. M. & Holbrook, J. ,I. (1977). In Pyridine A’ucleotide Dependent Hydrogertrrses (Sund, H.. Ad.), pp. 485-501, Walter de Gruyter, Berlin and New York. Plapp, B. V., Brooks, R. L. clr Shore, J. D. (1973). J. Riol. Ch,em. 248, 3470-3475. Plapp, B. V., Zeppezauer, E. & BrB;nditn, C-I. (1978a). J. Mol. Hiol. 119. 451b453. Plapp. B. V., Eklund, H. & BrSndBn, C.-I. (19786). J. Mol. Biol. 122, 23-32. Rao. S. T. Kr Rossmann, M. G. (1973). J. Mol. Rio!. 76. 241-256. Rossmann. $1. (:. & Blow, D. M. (1962). ilcta C’rystallogr. 15, 24.-31. Rossmann, M. G.. Liljas, A., Briindbn, C.-I. & Banaszak, L. J. (1975). In 2% El/zyrnPs (Boyer. I’. D., ed.), 3rd edit. vol. 11, pp. 61-102, Academic Press, New York. Sloan. I). I,., Young. .J. M. 8r Mildvan, A. S. (1975). Biochemistry. 14, 1998-2008. Subramanian. S. & Ross, P. D. (1978). Hiochemistry, 17, 2193-2197. Siidcrberg. B.-O.. Holmgren, A. Rr BrGndkn, C.-I. (1974). J. Mol. Riol. 90. 143~~152. Theorell. H. & (‘hance, B. (1951). Actu Chem. Scund. 5, 1127-I 114. tVhit,e. $I. L.. Hackert, M. L.. Buehner, M., Adams, M. J.. Ford, G. (‘.. Lentz, I’. .J.. Jr. Smiley. 1. E.. Steindel, S. ,J. 8r Rossmann. M. G. (1976). J. Nol. Biol. 102, 759-779. Zeppezauer. E., Siiderberg, B.-O., BrBndbn, C.-I., Akeson. A. B Theorell. H. (1967). Acts Phpm. Scnnd. 21. 1099~1101.