Biochimica et Biophysics Acta, 322 (1973) 124-132 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

B B A 365I 4

CHEMICAL AND STRUCTURAL RELATIONSHIPS OF NAD + AND PLATINUM BINDING TO MALATE DEHYDROGENASE

MICHAEL WADE", DEMETRIUS TSERNOGLOU"", EDWARD HILL""", LAWRENCE WEBB A.'~D LEONARD BANASZAK Department of Biological Chemislry, Washington University School of Medicine, St. Louis, NIo. (u.s .A.)

(Received March I3th, 1973)

SUMMARY

Platinum compounds have been shown by chemical studies to inhibit the catalytic activity of pig heart cytoplasmic malate dehydrogenase. Pseudo first-order rate constants for the inactivation are reduced by the presence of coenzyme. X-ray diffraction studies show that there are eight platinum binding sites in the crystalline state. These are paired between the two subunits, and the paired sites, for the most part, obey the molecular two-fold symmetry. Since the locations of the NAD ÷ binding sites are also known from the crystallographic analysis, the steric relation- ships between all pairs of platinum sites and the coenzyme binding site can be described. Two pairs of heavy metal sites taken singularly or together could account for the inhibition which occurs in solution.

I N T R O D U C T I O N

Malate dehydrogenase (L-malate: NAD oxidoreductase, EC 1.1.1.37), catalyzes the NAD+-dependent interconversion of L-malate and oxaloacetate. In the cells of many organisms, there are two separable forms of the enzyme; one localized in mito- chondria and one found in the cell cytoplasm or supernatant fraction. Both forms of malate dehydrogenase from mammalian heart muscle appear to be composed of two similar or identical subunits 1-~. The subunit molecular weight of cytoplasmic malate dehydrogenase is approx. 35 ooo (ref. 3)- The complete polypeptide conformation of cytoplasmic malate dehydrogenase from pig heart has been determined from an electron density map at 3.0 • resolution 4. The two polypeptide subunits are related in the three-dimensional structure by a two-fold rotation axis. This symmetry axis will be referred to throughout as the molecular dyad.

Abbreviations: I~ED, platinum ethylene diamine dichloride; PHMS, p-hydroxymercuri- phenyl sulfonate.

Present address : " Max-Planck-Institut ft~r Medizinische Forschung, Abteilung Biophysik, Heidelberg, Germany.

"* Department of Biochemistry, ~Tayne State University, Detroit, Mich., U.S.A. """ Department of Physiology, Vanderbilt University, Nashville, Tenn., U.S.A.

BINDING TO MALATE DEHYDROGENASE I 2 5

The method of multiple isomorphous heavy atom replacement was used in the structure determination. The heavy atom compounds found to bind to the cyto- plasmic malate dehydrogenase may be divided into three categories-mercurials, the uranyl ion, UO2 ~÷, and platinum compounds 5& By definition, an isomorphous derivative is one whose structure is not detectably different from that of the native enzyme. Hence changes in enzyme activity following isomorphous substitution must be largely due to binding at specific functional groups in the region of the active site.

Chemical studies to be described have shown that the platinum compounds used as derivatives are good inhibitors of cytoplasmic malate dehydrogenase. In order to compare binding sites in solution to those in the crystalline state, other factors must be considered. In the crystal, a binding site may include liganding groups from more than one enzyme molecule. Should this occur, that site would be energetically less favorable in solution. Furthermore, there is the possibility that a binding site exists in solution which is sterically blocked in the crystalline state by the packing of adjacent enzyme molecules. In spite of these two factors there should be a close correlation between the binding sites found in solution and those in protein crystals. In this study the effects of platinum compounds on the catalytic activity of cyto- plasmic malate dehydrogenase will be described and analyzed in terms of their proximity to the active site region in the crystalline state.

MATERIALS AND METHODS

All chemicals were reagent grade. NADH was purchased from Sigma Chemical Co. (St. Louis).

Pig heart cytoplasmic malate dehydrogenase was prepared by a modification of the method of Banaszak 7. The precipitation at pH 5 and acetone fractionation steps were omitted. Two types of crystalline habits of pig heart cytoplasmic malate dehydrogenase are known and are labeled A and C (ref. 5). Type A crystals are grown in the absence of NAD + at pH 6.2, while type C crystals are obtained in the presence of NAD + at pH 5. Type C crystals have been shown by Glatthaar et al. s to contain bound NAD + while no significant amount of NAD+ is found in type A crystals. All of the work to be described on crystalline cytoplasmic malate dehydrogenase was done on type C crystals. These were obtained by using large scale batch methods similar to the methods used for growing crystals for X-ray study 5. The crystallization is done in a 0.05 M acetate buffer, pH 5.0, which contains I mM EDTA and I mM dithiothreitol. The enzyme solution is brought to 60% saturation in ammonium sulfate and a IO-fold molar excess of NAD + to enzyme is added. The protein concen- tration at this point is about 5 mg/ml. Any turbidity is removed by centrifugation and a solution of saturated ammonium sulfate is added dropwise until a faint cloudi- ness persists. The enzyme solution is left at 4 °C and crystals generally begin to form within one week. Characterization of type C crystals is done by microscopic exami- nation of their habit 5 or by X-ray study.

In the preparation of heavy atom derivatives of the crystalline cytoplasmic malate dehydrogenase, the soaking solution contained 70% saturated ammonium sulfate in 0.05 M sodium acetate buffer at pH 5.0 to 5.1. The purity of redissolved type C crystals was checked by electrophoresis at pH 9-5, in 7.5% polyacrylamide gels using a discontinuous Tris-glycine buffer system. These gels had one band after

120 M. WADE et al.

staining for protein with aniline black or after staining for enzymatic activity ac- cording to the method of Goldberg 9.

The catalytic activity of cytoplasmic malate dehydrogenase was determined by following the rate of disappearance of NADH absorbance at 34 ° n m . These reactions were carried out at pH 7.4 in a solution which contained 50 mM Tris-HC1, o.I mM oxaloacetate and o.15 mM NADH.

The X-ray methods used in the study have been described in detail by Tserno- glou et al. 6. Computer display methods for studying the steric relationships of the platinum binding sites to the polypeptide chain of cytoplasmic malate dehydrogenase have been described in part by Jacobi et al. in the Appendix to ref. 4.

RESULTS AND DISCUSSION

Plat inum binding sites in the crystalline state X-Ray analysis using difference Patterson and difference Fourier methods

indicated that there are eight platinum binding sites in the asymmetric unit of type C crystals of cytoplasmic malate dehydrogenase n. Unpublished results showed that these sites are the same for both PtC14 2- and platinum ethylene diamine dichloride (PED). Since the asymmetric unit also contains one dimer of cytoplasmic malate dehydrogenase, there are 8 platinum binding sites per molecule of enzyme. After studying the positions of the bound platinums, it was immediately apparent that the binding sites are paired between the two subunits which comprise the molecule. The structural relationship between these binding sites and segments of the polypeptide chains will be described subsequently. The angular rotation, ~, by which one site must be rotated around the molecular dyad to bring it to the closest position to the related site in the other subunit is given in Table I. For a two-fold symmetry axis, T -- 18o °. Also shown in Table I is the distance of each site to the molecular dyad. The average disagreement between the paired sites is about :~ o.5 A. This small error is certainly well within the error anticipated from the accuracy of the present position

TABLE I

S Y M M E T R Y O F P L A T I N U M B I N D I N G S I T E S I N C R Y S T A L L I N E C Y T O P L A S M I C M A L A T E D E H Y D R O G E N A S E

The number ing scheme for the p la t imun sites and the two subuni ts of cytoplasmic malate dehy- drogenase is identical with tha t used in previous publications4, 6. The pairing of sites was obvious when the sites were displayed along with the a carbon model of the two polypeptide chains.r is the angle in degrees by which one PtCla 2- site mus t be rotated around the molecular dyad to bring it to its closest position to the equivalent site in the other subunit . For ideal two-fold rotat ional svmmetrv , v ~ 18o °. The perpendicular distance from each site to the molecular dyad is given in ~,~, The posit ion of the molecular dyad was determined from the a carbon model at 3.0 ~ resolu- tion a.

Pair r (o) Subuni t z Subuni t 2

Pla t inum Distance to site dyad ( d )

l 18i Pt-1 2 8 . i [[ I75 Pt-3 ~2.3

1I[ 195 Pt-8 16.1 IV i8 i Pt-5 19.4

Pla t inum Distance to site dyad (A )

I ' t-2 26. 7 Pt-6 io.o Pt- 7 16.2 Pt- 4 18.8

B I N D I N G TO M A L A T E D E H Y D R O G E N A S E 12 7

of the local dyad 4. In solution, the b ind ing of small molecules to a dimer of ident ical polypeptide subuni t s should, under most conditions, result in pairs of identical sites. The results shown in Table I indicate tha t this proper ty is also found in the crystall ine state of cytoplasmic malate dehydrogenase.

The effect of p l a t inum compounds on cytoplasmic malate dehydrogenase ac t iv i ty was tested by reacting crystals under condit ions identical to those used in the X-ray analysis. The subs t i tu ted crystals were then washed free of excess PtC1, 2- or P E D solution, redissolved and assayed for enzymat ic activity. As controls, batches of both the nat ive crystals and crystals soaked in p -hydroxymercur iphenyl sulfonate (PHMS) were t reated in the same way.

The effects of the heavy metals on cytoplasmic malate dehydrogenase ac t iv i ty are shown in Table II . The catalyt ic ac t iv i ty of cytoplasmic malate dehydrogenase obtained from P E D or PtC142--substituted crystals is essentially zero. Crystallo- graphic studies have shown tha t three molecules of PHMS are bound per molecule of cytoplasmic malate dehydrogenase 6, and Table I I indicates tha t this reaction has no detectable effect on the activity. The PHMS sites are known to be at different locations than the p l a t inum sites in the crystall ine enzyme 6.

TABLE II

THE EFFECT OF HEAVY ATOM COMPOUNDS ON THE ENZYMATIC ACTIVITY OF CYTOPLASMIC MALATE DEHYDROGENASE IN THE CRYSTALLINE STATE Native crystals were washed twice with a solution of 70% ammonium sulfate in 0.05 M sodium acetate, pH 5.1, prior to soaking in the heavy atom solutions. The soaks, containing 1. 5 mg of cytoplasmic malate dehydrogenase, were allowed to stand for about one week at 4 °C. Both the platinum compounds and the PHMS were made to the indicated concentrations in the ammonium sulfate-acetate buffer. The soak time is based on X-ray measurements although most of the bind- ing is complete in 48 h. At the end of the incubation, the crystals were washed once with the am- monium sulfate-acetate buffer, redissolved in 0.05 M Tris-HC1, pH 7.0 and assayed for enzymatic activity in triplicate. The % activity remaining is based on a control of native crystals.

Heavy atom Concn (mM) % activity compound remaining

PtC142- 0. 5 0.5 PED 6. 5 (satd soln) 1. 7 PHMS 0.5 IOI .o

The reaction of platinum compounds with cytoplasmic malate dehydrogenase in solution As expected, the inhibi t ion of cytoplasmic malate dehydrogenase by p la t inum

compounds occurred rapidly in solution. However, the rate of inhib i t ion varied when substrates were added. The results of the experiments to determine the rate of inhi- bi t ion and the effect of substrates are summarized in Table I I I . Because the p la t inum compounds are present in large excess over the cytoplasmic malate dehydrogenase, first-order kinetics were observed for the inhibi t ion reaction near 0.5 mM PtC142-. At higher concentrat ions the rates did not appear to follow first-order kinetics. The lower rate cons tan t observed for P E D is perhaps the result of a l igand exchange reaction which occurs during the b inding process. This reaction would be different for the two forms of the p la t inum.

As can be seen in Table I I I , the presence of either NAD+ or N A D H significantly reduced the rate of inact ivat ion. No significant effect on the rate cons tant of inac-

1 2 8 M. W A D E et al.

T A B L E I I I

P S E U D O F I R S T - O R D E R R A T E C O N S T A N T S F O R P L A T I N U M I N H I B I T I O N O F C Y T O P L A S M I C M A L A T E D E H Y -

D R O G E N A S E A C T I V I T Y

T h e p s e u d o f i r s t - o rde r r a t e c o n s t a n t k w a s d e t e r m i n e d in 0.05 M Tr i s -HCl , p H 7.0 a t 23 °C in t h e p r e s e n c e a n d a b s e n c e of c o e n z y m e or s u b s t r a t e . All so lu t ions h a d a f inal e n z y m e c o n c e n t r a t i o n o f 0 .03 /zM. T h e r e a c t i o n s w e r e s t a r t e d b y t h e a d d i t i o n o f c y t o p l a s m i c m a l a t e d e h y d r o g e n a s e to t h e p l a t i n u m so lu t ions . S a m p l e s w e r e w i t h d r a w n a t a b o u t f ive i n t e r v a l s a n d a s s a y e d for c y t o p l a s m i c m a l a t e d e h y d r o g e n a s e a c t i v i t y as d e s c r i b e d in t h e t e x t .

Heavy atom Conch Substrate Concn k (rain -1) compound (raM) (mM)

PtC14~- o. I - - o.o9 PtC14 ~- o.5 - - o.21 PtC142- I .o - - o.23 P E D I.O - - o.o23 PtC142- o. 5 N A D ~ o. 5 o. 13 PtC14~- 0. 5 N A D H 0. 5 o.o41 PtC142- 0. 5 m a l a t e 0. 5 o,2 i PtC142- o.5 o x a l o - a c e t a t e o.5 o,21

t ivation was observed in the presence of o.5 mM L-malate or o.5 mM oxaloacetate. The reversibility of the platinum binding to cytoplasmic malate dehydrogenase

was first checked by gel filtration. Twenty #1 of a crystalline suspension of cyto- plasmic malate dehydrogenase were added to o.2 ml of a o.o2 M K2PtC14, o.o5 M Tris-HC1, pH 7.° solution. As a control, a sample of cytoplasmic malate dehydrogenase was treated in the same way but the PtC142- was omitted. When i % of the control enzymatic activity remained, the enzyme-PtC142- solution was applied to a o. 9 cm × 7 ° cm column of Sephadex G-25 equilibrated with the Tris buffer. The effluent from the column was monitored at 24 ° nm where both cytoplasmic malate dehydrogenase and K@tC14 absorb. The control enzyme solution and the PtC142- were also chro- matographed under identical conditions. The elution profile of the K2PtC14-treated enzyme showed two well separated peaks of material absorbing at 24o nm. The first peak appeared in the void volume as did the cytoplasmic malate dehydrogenase from the control enzyme solution, while the second peak eluted in the same position as PtC142-. This fraction of cytoplasmic malate dehydrogenase reacted with PtC142- contained less than I °/o of the enzymatic activity of the corresponding fraction from the untreated enzyme.

The absorption spectra after gel filtration of native cytoplasmic malate de- hydrogenase, the cytoplasmic malate dehydrogenase-PtC142- complex, and 1.6. IO 4 M K2PtC14 is shown in Fig. I. I t is evident that even after gel filtration some of the PtC142- remained bound to the enzyme. This confirms the results of the activity measurements. The cytoplasmic malate dehydrogenase-PtCi42- complex is identi- fiable by its ultraviolet absorption spectrum, the complex having a higher absorbance than native cytoplasmic malate dehydrogenase in the 24o-26o nm region. Some of these differences could be due to the presence of small amounts of denatured enzyme. However, this would seem unlikely in view of the small but definite differences, in the 3oo-32o nm region of the spectra of cytoplasmic malate dehydrogenase-PtC142 com- plex compared with the native cytoplasmic malate dehydrogenase. This is also where PtC14 ~- alone absorbs weakly. One further a t tempt to reverse the platinum inhibition by dialysis indicated only a small return of cytoplasmic malate dehydrogenase

B I N D I N G TO MALATE D E H Y D R O G E N A S E 12 9

; o 0.3 ; ~

~o.2 o ~ -

\ '..,/ k\

I I I I I 240 260 280 300 320 w a v e l e n g t h - nm

Fig. i. Adsorption spectra of cytoplasmic malate dehydrogenase and PtC142- complex. The ab- sorption spectra are of pooled samples after Sephadex G-25 chromatography as described in the text. The solid line is the spectrum of the cytoplasmic malate dehydrogenase-PtC142- complex. The dashed curve is for cytoplasmic malate dehydrogenase and the dotted curve is the spectrum of PtC142-.

activity. The cytoplasmic malate dehydrogenase activity increased from 6 to 16% of the control after 15 h of dialysis against multiple changes of 0.03 M potassium phosphate, I mM EDTA, pH 7.0.

The results obtained by the studies using PtC142- resemble the inhibition of cytoplasmic malate dehydrogenase by iodoacetate described by Leskovac and Pfleiderer 1°. They found that pig heart cytoplasmic malate dehydrogenase reacted with iodoacetate to alter an average of one methionyl residue per subunit resulting in the irreversible inhibition of the enzyme. The rate constant for inactivation decreased 1.3- and 1.8-fold in the presence of NAD + and NADH (I raM), respec- tively 1°. Oxaloacetate and L-malate at a concentration of o.I M did not affect the inhibition 1°. Leskovac and Pfleiderer found that the inhibited enzyme still would bind NADH. However, either some of the binding sites for the coenzyme were com- pletely blocked or the dissociation constant was increased 1°. The rate constants for the inhibition of cytoplasmic malate dehydrogenase by PtC14 ~- and the effect of substrates as shown in Table I I I are qualitatively similar to those described for iodo- acetate 1°. Furthermore, in practically all of the cases where PtCI42- has been used in the structural analysis of crystalline proteins, a methionyl side chain was involved in the platinum binding site 11.

Recent studies on the structure of cytoplasmic malate dehydrogenase at 2.5 resolution have described the location of the NAD + binding site (Webb, L., Hill, E. and Banaszak, L. J., unpublished). I t is, therefore, possible to show the steric relationship of the platinum binding sites to the active site of the enzyme in the crystalline state. Figs 2A and 2B are stereo views of the a carbon atoms of the two subunits of cytoplasmic malate dehydrogenase with the platinum sites and the stick model of the bound NAD+. The orientation of NAD+ was displayed directly from coordinates obtained from an optical model building device without any refinement of bond lengths, bond angles or torsional angles ~2. These coordinates are being refined

I3O l~I. WADE ef al.

and the conformation of the bound coenzyme studied in detail. While the torsional angles may change slightly from those shown in Figs 2A and 2B, the position of the coenzyme will not change noticeably.

I t is readily apparent from Figs 2A and 2B that all of the platinum binding sites are on the surface of the molecule, well separated from the polypeptide chain. The amino acid side chains which are responsible for binding are still undetermined. I f one looks at the folding of polypeptide chain near each platinum site, the structural pairing as described in Table I is also apparent.

The binding sites labeled Pt- 4 and Pt-5 are near the subunit interface and are

-O5

I " f ' t /

B

A .e-5

q

5

4O-

5

4-e-

Fig. 2. S tereoview of the a ca rbon model of cy top la smic ma la t e dehydrogenase . The figure m u s t be v iewed wi th a set of stereo glasses. Dis tances m a y be e s t ima ted using the fact t h a t an a carbon to a carbon connect ion is 3.8 ~. The p l a t i n u m sites are m a r k e d wi th b l ack dots and n u m b e r e d as in Table I and in an earl ier pub l i ca t i on 4. The local d y a d is labe led D and the ca rboxy- and amino- t e rmina l s are labe led C and N, respect ive ly . Fig. 2A is of the s u b u n i t labe led I in Table I and 2B is s u b u n i t 2. The NAD + can be seen s l igh t ly to the left of Pt-3 in Fig. 2A and to the r igh t of P t -6 in Fig. 2B. As an aid to loca t ing the N A D + molecules in the a carbon model, the symbol A is near the adenine end of the N A D + bound to Subun i t i . The symbo l A ' is p laced near the cor responding region in Subun i t 2. The s t r u c t u r a l r e la t ionsh ip of the two subun i t s in the comp]ete molecule can be surmised by super impos ing the dyad, D in Fig. 2A and 2t3.

B I N D I N G TO M A L A T E D E H Y D R O G E N A S E 131

not dose to the NAD + binding site. I t is difficult to see how substitution at these sites would affect the active sites. As can be seen in Figs 2A and 2B, the paired sites P t - i and Pt-2 are in the general region of the adenine end of the NAD + binding site. But again they are far enough away that it does not seem likely that they could interact with any atoms in the active sites.

There are, therefore, two pairs of crystallographic sites which could be related to the inhibition of cytoplasmic malate dehydrogenase in solution. They are the pairs Pt- 7 and Pt-8, and Pt-3 and Pt-6. The symmetry related sites Pt-3 and Pt-6 are very close to the adenine ribose region of the NAD + binding site, as can be seen in Figs 2A and 2B. These platinum sites are positioned on the solvent side of the cleft that constitutes the NAD+ site. They appear to be located such that interference with the binding and dissociation of NAD + is possible.

The symmetry related sites, Pt- 7 and Pt-8 also are relatively close to the NAD + binding site. They are separated from the nicotinamide end of the binding site by a segment of extended polypeptide chain. This segment makes a turn into a helical region (/~DaD turn in ref. 4). This turn or "loop" of polypeptide chain in the homo- logous dehydrogenase, dogfish LDH, moves about 12 A in the ternary complex to close the entrance to the NAD + cleft 13. I f a homologous conformational change occurs in cytoplasmic malate dehydrogenase, and if this change is necessary for enzymatic activity, the platinum binding sites at Pt- 7 and Pt-8 might interfere with the catalytic reaction. In the crystalline state, the occupancy at Pt-8 is considerably lower than at the other binding sites 6. The structural basis for this reduced substi- tution is not yet known. I t is important to emphasize that while the cytoplasmic malate dehydrogenase molecule has been shown to have approximately two-fold symmetry, subtle conformational differences may exist between the two subunits 4. This could explain the difference in occupancies at sites Pt- 7 and Pt-8. An exact comparison of the two subunits will not be possible until the complete amino acid sequence and the coordinates of all atoms in the two polypeptide chains are known.

Qualitatively, it would seem that platinum compounds bind to both the apo form of cytoplasmic malate dehydrogenase and to the enzyme with bound NAD +. The platinum complexes of cytoplasmic malate dehydrogenase are inactive. In so- lution, the first-order rate constants for the PtC14 ~- inactivation are reduced by the presence of NAD+ or NADH. Two pairs of PtC142--binding sites, Numbers I I and I I I in Table I, are near the NAD+-binding sites of cytoplasmic malate dehydrogenase in the crystalline state. The other four sites, Pairs I and IV in Table I, appear to be too far from the active site regions to account for the inhibitory effects observed in solution. The four platinum sites comprising Pairs I I and I I I , taken singularly or together, could explain the inhibition results.

ACKNOWLEDGEMENTS

The technical assistance of Mr G. Barbarash and Miss B. Stadtmiller is grate- fully acknowledged. This work was supported by grants from the National Science Foundation (GB-27437), National Insti tutes of Health (GM-I3925) and the Life Insurance Medical Research Fund. Edward Hill was a postdoctoral fellow of the National Insti tutes of Health (I-FO2-GM49896) ; M. J. Wade is grateful for support from the PHS Training Grant (GM-714) ; and L. J. Banaszak holds a Research Career

132 M. WADE et al.

D e v e l o p m e n t A w a r d (GM-I4357) . W e a re i n d e b t e d to C. D. B a r r y , J . M. F r i t s c h ,

a n d T. J a c o b i w h o are r e s p o n s i b l e for t h e d e v e l o p m e n t of t h e m o l e c u l a r d i s p l a y

s y s t e m 4 a n d for t h e i r c o n t i n u e d he lp in t h e a p p l i c a t i o n of t h i s s y s t e m to t h e s t r u c t u r a l

a n a l y s i s of e n z y m e s .

R E F E R E N C E S

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Macromolecules and Membranes (Chance, B., Yonetani, T. and Mildvan, A., eds), Vol. 2, pp. 71-8o, Academic Press, New York

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mun. 46, 757-765 9 Goldberg, E. (1962) Science 139, 602-603

io Leskovac, V. and Pfleiderer, G. (1969) Hoppe-Seyler's Z. Physiol. Chem. 35 o, 484-492 I I Dickerson, R., Eisenberg, D., Varnum, J. and Kopka, M. L. (1969) J. Mol. Biol. 45, 77-84 12 Richards, F. M. (1968) J. Mol. Biol. 37, 225-23 ° 13 Adams, M. J., Ford, G. C., Koekoek, R., Lentz, P. J., McPherson, A., Rossmann, M. G.,

Smiley, I. E., Schevitz, I~. W. and Wonacott , A. J. (197 o) J. Mol. Biol. 227, lO98-11o3