The structure of dehydrogenases

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227

37 Grafius, M. A. and Millar, D. B. (1965) Biochim. Biophys. Acta 110, 540-547 38 MassouliC, J. and Rieger, F. (1969) Eur. J. Biothem. 11,44-455 39 Massoulit;J., Rieger, F. and Bon, S. (1971) Eur. J. Biochem. 2 l,542-55 1

40 Rieger, F., Bon, S., Massoulit, J. and Cartaud, J. (1973) Eur. J. Biochem. 34, 539-547 41 Bon, S., Rieger, F. and MassouliC, J. (1973) Eur. J. Biochem. 35,372-379 42 Soucie, W. G., Voss, H. F. and Wilson, I. B. (1975) Engineering Foundation Conference on Enzyme Engineering, Portland, Ore., 1975 (in the press) 43 Rash, J.E. and Ellisman, M. H. (1974) J. Cell. Biol. 63, 567-586 44 Kefalides, N.A. (1973) Ini. Rev. Corm. Tissue Res. 6,63-104 45 Rieger, F., Bon, S. and Massoulit, J. (1973) FEBS Lett. 36, 12-16 46 Taylor, P., Lwebuga-Mukasa, J., Berman, H. and Lapi, S. (1975) Croat. Chim. Acta 47, 251-263

47 Anglister, L. and Silman, I. (1975) to be published 48 Piez, K.A. and Gross, J. (1959) Biochim. Biophys. Acta 34, 24-39 49 Anglister, L., Rogozinski, S. and Silman, I. (1976) Abstracts, Annual Meeting Israel Biochem. Sot.,

(LADH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). There is also reasonable evidence that NADP-dependent glutamate dehydrogenase contains two such domains. In contrast, the structure of the catalytic domain is, in general, different for each dehydrogenase.

April, p. 76 50 Reid, K.B. M. and Porter, R. R. (1975) in Contemporary Topics in Molecular Immunology, pp. l-22, Vol. 4 (Inman, F. P. and Mandy, W. J., eds), Plenum Press, New York 5 1 Cartaud, J., Rieger, F., Bon, S. and Massoulii, J. (1975) Brain Res. 88, 127-130 52 Livne, A. and Bar-Yaakov, 0. (1976) Biochim.

Common co-factor binding domain

Biophys. Acta 419, 358-364 53 Wilson, B. W. and Walker, C. R. (1974) in Exploratory Concepts in Muscular Dystrophy-II, pp.

136-153, (Milhorat, A.T., ed.), Excerpta Medica, Amsterdam

The structure of dehydrogenases William Eventoff and Michael G. Rossmann Recent three-dimensional determinations ofvarious dehydrogenase structures have shown some common architectural and chemical principles. This has provided insights into such diverse problems as catalysis, evolution andprotein folding. Introduction Dehydrogenases are among the most fundamental biological catalysts as they are essential in the reduction 6f substrate in the process of obtaining energy in all cells. The overall reaction can be represented by DH2+A-+D+AH2

(I)

where DH, is a substrate and A is an intermediate hydrogen carrier. The final product of a series of such linked reactions can be water and energy. The individual members of this class differ in their requirements for metal ions, the stereospecificity for the hydride transfer catalyzed, and the nature of the intermediate hydrogen carrier. The latter is usually a nucleotide such as FMN, FAD, NAD or NADP. Since the pioneering work of Wieland, Warburg, von Euler, Keilin and others in the early part of this century, biochemical investigations have generally tended to demonstrate the considerable chemical and physical differences of the many known dehydrogenases. Recently, however, the three-dimensional structures of four different NAD linked dehydrogenases combined with amino acid sequence data (Table I) have made it W.E. andM.G.R. are at the Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, U.S.A.

possible to discern some of their basic common principles. References to original sequence and structural contributions will be found in Table I. It has been found that the long polypeptide chains of large proteins mostly fold into a series of spatially separated domains. All the dehydrogenase structures studied to date have been found to consist of essentially two domains with different functions. One of these domains bitids the coenzyme whereas the other binds the substrate, determines specificity, and provides many of the residues needed for catalysis. These have been called the NAD binding and catalytic domain, respectively. Although the position of the former domain within the total polypeptide chain varies (Fig. l), it has approximately the same structure in lactate dehydrogenase (LDH), soluble malate dehydrogenase (sMDH), liver alcohol dehydrogenase 6

B

D

22

A,

,

Al

_

196 Al AI

_

The coenzyme binding domain (Fig. 2) consists of a six-stranded parallel pleated sheet @A, pB, PC, /3D, BE, /?F) and four helices (aB, crC, crE, alF). Starting from the amino end of the sheet, the first structural element is PA. Helices aB and crC form the connections between /3A and /?B and BB and /3C, respectively, and are on the same side of the sheet. This threestranded parallel p structure forms the AMP binding unit. From /3C, the polypeptide chain passes back to the amino end of the sheet and lays down /?D next to /3A. The sequence /3D, aE, @E, alF, PF forms the NMN binding unit. Rao and Rossmann [l] showed that there is a twofold axis approximately parallel to /?A and PD relating these two mononucleotide binding units. The coenzyme binds in an open conformation. The adenine binds in a non-specific hydrophobic pocket at the carboxy end of the parallel pleated sheets between strands BB and PD. The non-reactive side of the nicotinamide ring also appears to be in a hydrophobic environment, in contrast to the hydrophilic surroundings of the reactive side of the ring. In addition to these contacts, the coenzyme is further oriented by a number of hydrophilic contacts which include interactions of both polar main chain atoms and amino acid side chains with the riboses and pyrophosphate linkage. Lactate dehydrogenase Lactate dehydrogenase was the first dehydrogenase to have its structure determined. The enzyme is a tetramer and has a molecular weight of 140,000. Two different types of subunits, coded for by independent gene loci, are found in most tissues. The M (or A) type subunit is the predominant form in anaerobic tissue, such as skeletal muscle; and the H (or B) subunit is the form predominant in aerobic

A2

165

C

A2

Y

F

A2

316

A2

144

E

329

LDH 3y

376

GAPDH LADH

c_

307

s-MDH

Fig. I. Position of the dinucleotide binding domain A within the polypeptide chain of various dehydrogenases. The different catalytic domains are labeled B, C, D,. .. The catalytic domains of C of LDH and s-MDH have the same structure. The nucleotide binding domain is itself the repetition of two similar mononucleotide binding structures related by an approximate two-fold axis.

228

TIBS - October

1976

TABLE I Properties of some dehydrogenases Enzyme

Lactate dehydrogenase Glyceraldehyde-3phosphate dehydrogenase Liver alcohol dehydrogenase Soluble malate dehydrogenase Mitochondrial malate dehydrogenase Yeast alcohol dehydrogenase

Abbreviation

Function

Amino acid sequence

Review ref.

Threedimensional structure

Date

Ref. ___-

Date

Ref.

Co-factor

NAD speciticity

Number subunits per molecule

of

Essential metal atoms

Mol. wt.

LDH

glycolysis

1973

14

1970

15

2

NAD

A

4

none

140,000

GAPDH

glycolysis

1967

16

1973

17

18

NAD

B

4

none

144,000

1970

19

1973

20

4

NAD

A

2

Zn

84,000

1972

21

22

NAD

A

2

none

72,000

cf. 23

NAD

A

2

none

72,000

1973

3

NAD

A

4

none

160,000

1972

24,25

NADP

B

6

none

336,000

1975

26

NAD

?

4

none

464,000

LADH s-MDH

alcohol metabolism citric acid cycle

m-MDH

citric acid cycle

YADH

glycolysis

Glutamate dehydrogenase

GluDH

Glutamate dehydrogenase

GluDH

amino acid biosynthesis energy supplying catabolism

tissue, such as the heart. The association of these subunits to form the LDH tetramer gives rise to five isoenzymes, H,, H,M, H,M,, HM, and M,; the distribution of which is tissue dependent. The M, isoenzyme from dogfish skeletal muscle has been studied extensively by Xray crystallography. The structures of the co-factor free (apo), the co-factor bound (holo) and some co-factor-inhibitor complexes of the enzyme have been studied for a variety of isoenzymes. These different enzymatic states provide an opportunity for observing different steps in the cataly-

tic process. When these results are comIbined with the amino acid sequence (deter-

mined by Dr Susan S. Taylor at the University of California, San Diego), it is possible to determine the chemical triggers of the conformational changes essential to nucleotide and substrate binding prior to xtalysis as shown in Fig. 3 and explained more fully by Holbrook et al. [2]. ,Soluble malate dehydrogenase

In 1972, the three-dimensional structure 3f a binary NAD+ complex of pig heart +MDH revealed that both the overall

folding of the polypeptide chain and the position and orientation of the bound nucleotide co-factor were very similar to that observed in LDH. Thus, with the exception of one small part, both the NAD binding domain and the catalytic domain are the same in LDH and s-MDH. The most sifnificant difference in the two structures is the absence of the first 20 residues of LDH in s-MDH (Fig. 1). These residues form an ‘arm’ which extends from the rest of the subunii styucture which itself is more compact and globular. This region in LDH is essential for the formation of the tetramer and its absence in s-MDH must be the reason why only a dimeric structure is formed. The primary structure of s-MDH is not known and consequently the details of the interactions between the protein and the coenzyme and substrate are also not available. In spite of this, in view of the tertiary and quaternary structural homology between the two dehydrogenases and the similarity in the orientation and conformation of the bound NAD+ co-factor, it seems reasonable to speculate that they may have diverged from a common precursor only slightly before the divergence of the LDH isoenzymes and, furthermore, that amino acid substitutions in the vicinity of the active site have led to their different specificities. Alcohol dehydrogenase

111 CO0t-i

Fig. 2. Schematic diagram of nucleotide binding domains. (Taken from ref. [9/ with permission.)

Both lactic acid and alcohol can be the end product of anaerobic glycolysis. The last step in this process is controlled by lactate or alcohol dehydrogenase, respectively. The amino acid sequence of yeast alcohol dehydrogenase (YADH) has been

229

Fig. 3. Schematic diagram of the LDH active site. It shows bound NAD forming on odduct with pyruvaie to form an abortive ternary complex enzyme-coenzyme-substrate. The coenzyme is guided into its position on the enzyme by initially finding the hydrophobic pocket for the odenine. This then attracts orginine IOI, causing a large conformational change which finishes creating the substrate binding site with arginine 109. Once the lactate substrate has bound as in an active complex, it can then donate a proton to histidine I95 and a hydride to the coenzyme. Asymmetr), of the site excludes D-lactate as a substrate.

studied by Jijrnvall [3] who showed it to be largely homologous to liver alcohol dehydrogenase. The structure of yeast alcohol’ dehydrogenase (a tetramer) has not yet been determined. Although the physiological function of liver alcohol dehydrogenase (LADH), whose structure is known, is not fully understood, its homology to YADH must indicate that the folding and mechanism of the two enzymes is very similar. Horse LADH is a dimer. of molecular weitht 84,000 which containstwo Zn2+ per subunit. The mode of association between the two,identical subunits of LADH differs from that observed in LDH and s-MDH. The catalytic domain consists of two parts: the first 175 and the iast55 residues.

The two ZN2+ are contained in the catalytic domain, although they have different functions. One of them is required for maintaining the overall folding of the enzyme and has been termed the structural zinc. It is bound in a loop, formed by residues 95-113, which projects out from the catalytic domain and has few side chain interactions with the remainder of the structure. The coordination sphere is composed of four cysteine residues, 97, 100, 103 and 111, arranged in a distorted tetrahedron and is similar, both with respect to composition and geometry, to that found for the iron ion in rubredoxin. Furthermore, the distribution of coordinating cysteines in the amino acid sequenceand the hand of the coordination

sphere is similar to that found in bacterial ferredoxin. Branden et al. [4] have suggested that thiseither represents an energetically favorable arrangement or has evolutionary implications. The second zinc, which is essential for catalysis, is bound in the bottom of the active site pocket. It is also tetrahedrally coordinated, although the coordination sphere, in contrast to the structural zinc, is heterogeneous being composed of two cysteines, 46 and 174, a histidine, 67, and either a water molecule or hydroxide ion. Model building studies have indicated that an alcoholate ion can bind directly to the Zn* +, in the apo enzyme structure, by displacing a water molecule in such a way as to position the H atom to be transferred close to the A-side of the nicotinamide ring. The conformation of the complete coenzyme has only recently been studied by X-ray diffraction (C. I. Brand& private communication) and confirms the position of the nicotinamide ring as deduced from the conformation of the ADP-ribose in the binary complex with analogy to the structure of NAD + in LDH and s-MDH. On the otherahand, Sloan et al. [5] have proposed, based on NMR studies of two ternary inhibitor complexes LADHLADHNADH-isobutyramide and NADH-ethanol, of a Co*+ containing enzyme that there is no direct interaction between the ZnZ + and substrate. They propose instead that an outer sphere complex is formed in which the water of hydroxyl group bound to the zinc interacts with the substrate. Crystals of the ternary complex LADH-NADH-isobutyramide, the same complex as used in the NMR studies, have been grown and the solution of this structure by X-ray diffraction will undoubtedly resolve this discrepancy (C. I. Brand&, private communication). Glyceraldehyde-3-phosphate dehydrogenase

Fig. 4. Possible steps in the formation of a thioester intermediate during the oxidation-reduction step catalyzed by GAPDH. A subsequent phosphorylation and cleavage of the stable intermediate shown in (d) gives rise to the product I,3_diphosphoglyeerote.

The three-dimensional structure of the holo form of lobster tail GAPDH was determined in 1973. The tetrameric enzyme has a molecular weight of 144,000. The major difference between the conformation of the NAD + in the present structure and that found in the other dehydrogenases is the orientation of the nicotinamide ring. In GAPDH it is rotated approximately 180” about the glycosidic bond with respect to the other structures, and reflects the fact that the B-side of the nicotinamide ring, as opposed to the Aside in LDH, s-MDH and LADH, is used for the hydride transfer in this enzyme. The structural homology of the NAD binding domains in dehydrogenases thus shows that the divergence of A- and B-side

230 enzymes occurred after the development of a general structure for the binding of NAD. As in the case of LADH, the catalytic domain of GAPDH shows no structural relationship to the other dehydrogenases. The function of this enzyme is two-fold, namely a dehydrogenation followed by a phosphorylation. The positions of the substrate and inorganic phosphate, required for the two reactions, have been deduced from sites occupied by anions in the structure of the holo enzyme. Moras et al. [6] have used this information in conjunction with available biochemical kinetic data to suggest a possible mechanism for the catalytic process (Fig. 4). Another major difference between GAPDH and the other dehydrogenases is the mode of association of the subunits. Whereas in LDH, s-MDH and LADH the active sites are well separated, on the surface of the molecule, in GAPDH the active sites are close to the molecular center. This close proximity of active sites may represent a means of communication between subunits and thus be a possible explanation for the cooperative phenomenon displayed by this dehydrogenase. Evolution of the dehydrogenases The similarity in the overall folding of the nucleotide binding domains together with the obvious differences in the catalytic domains and quaternary structures of LDH, s-MDH, LADH and GAPDH led Buehner et al. [7] to postulate a scheme for their evolution (Fig. 5). Rossmann et al. [8] and Ohlsson et al. [9] aligned the .amino acid sequences by using the structural homology and they were able to show that the residues which are essential for the binding of the nucleotide co-factor are conserved. Using this approach, Rossmann et al. [8] and Eventoff and Rossmann [lo] were able to quantitate the original evolutionary suggestions of Buehner et al. [7]. An extension of these ideas to a protein of unknown three-dimensional structure is the demonstration of the two nucleotide binding domains in glutamate dehydrogenase (GluDH) from the primary structure alone. Wootton [ 1l] applied Chou and Fasman’s [ 12,131 rules for folding to the primary structure of both bovine and Neurospora GluDH. From the predicted regions of helices and sheets he located two regions in each enzyme which had the alternating sheet and helix structure common to the nucleotide binding domains previously described. He then used a variety of criteria, including the conservation of the structurally invariant and functionally essential residues, to align the amino acid sequences of both GluDHs

TIBS - October

with the other dehydrogenases. One of these alignments had indeed been previously recognized by Rossmann et al. [8] using a different approach. The picture which emerges from these studies is one in which a primordial nucleotide binding protein duplicated and became fused with a number of different catalytic domains. The nucleotide binding domain has also been found in flavodoxin and two kinases, phosphoglycerate kinase and adenylate kinase. Conclusions Although the details of the catalytic mechanism in dehydrogenases are still incomplete, some common features have emerged. The coenzyme is bound in an extended conformation; the nicotinamide is correctly positioned by hydrophobic interactions on the side away from the substrate and by hydrophilic interactions on the reactive side; and conformational changes accompany the formation of an active complex. Above all, the unexpected similarity in the structures of the dehydrogenases has provided the major impetus for the development of new methods for the comparison of protein structures. For instance, these have been successfully employed in demonstrating structural homology between cytochrome 6, and myoglobin and between T4 phage and hen egg white lysozyme. Since the three-dimensional structure of proteins can be conserved for a longer period of time than the recognizable similarity in amino acid sequence, this new field of molecular taxonomy will yield information on the evolution of protein families and consequently the important functional residues in the various conserved domains. In addition, for the same reasons, it can also be expected to shed some light on the nature of the folding mechanism in polypeptides, a subject which to date, although many theories have been put forth, has defied understanding.

Fig. 5. Evolutionary scheme for the evolution of dehydrogenases and their relation to flavodoxin as suggested by Buehner et al. [7/. (Taken from ref [7] with permrssron. J

I976

Acknowledgments We are grateful to Sharon Wilder for help in the preparation of the manuscript and to grants from the National Institutes of Health (no GM 10704) and the National Science Foundation (no. BMS74-23537). References 1 Rao, S. T. and Rossmann, M. G. (1973) J. Mol. Biol. 76, 241-256 2 Holbrook, J.J., Liljas, A., Steindel, S.J. and Rossmann, M. G. (1975) in The Enzymes (Boyer, P.D., ed.) Vol. XI, 3rd Edn, pp. 191-292, Academic Press, New York 3 Jornvall, H. (1973) Proc. Nar. Acad. Sci. U.S.A. 70, 229552298 4 Brlnden, C. I., Jiirnvall, H., Eklund, H. and Furugren, B. (1975) in The Enzymes (Boyer, P. D., ed.), Vol XI, 3rd Edn, pp. 103-190, Academic Press, New York 5 Sloan, D.L., Young, M. J. and Mildvan, A.S. (1975) Biochemistry 14, 199882008 6 Moras, D., Olsen, K. W., Sabesan, M.N., Buehner, M., Ford, G.C. and Rossmann, M.G. (1975) J. Biol. Chem. 250,9137-9162 7 Buehner, M., Ford, G.C., Moras, D., Olsen, K. W. and Rossmann, M.G. (1973) Proc. Nat. Acad. Sci. U.S.A. 70,3052-3054 M.G., Moras, D. and Olsen, K. W. 8 Rossmann, (1974) Nature 250, 194-199 B. and Branden, C.I. 9 Ohlsson, I., Nordstrom, (1974) J. Mol. Biol. 89,339-354 M.G. (1975) in 10 Eventoff, W. and Rossmann, CRC Critical Reviews in Biochemistry (Fasman, G. D., ed.), pp. 11 l-140, CRC Press, Cleveland 11 Wootton, J. C. (1974) Nature 252, 542-546 12 Chou, P. Y. and Fasman, G. D. (1974) Biochemistry 13,211&221 13 Chou, P. Y. and Fasman, G. D. (1974) Biochemistry 13, 222-245 14 Taylor, S. S., Oxley, S. S., Allison, W. S. and Kaplan, N. 0. (1973) Proc. Nat. Acad. Sri. U.S.A. 70, 179%1794 K., 1.5 Adams, M.J., Buehner, M., Chandrasekhar, Ford, G.C., Hackert, M. L., Liljas, A., Rossmann, M. G., Smiley, I. E., Allison, W. S., Everse, J., Kaplan, N.O. and Taylor, S.S. (1973) Proc. Nat. Acad. Sci. U.S.A. 70, 19681972 16 Jones, G. M. T. and Harris, J. I. (1972) FEBS Lett. 22, 185189 17 Buehner, M., Ford, G.C., Moras, D., Olsen, K. W. and Rossmann, M. G. (1974) J. Mol. Biol. 90,2549 18 Harris, J. I. and Waters, M. (l976)in The Enzymes (Boyer, P. D., ed.), 3rd Edn, Academic Press, New York (in the press) 19 Jornvall, H. (1970) Eur. J. Biochem. 16, 41-49 20 Eklund, H., Nordstrom, B., Zeppezauer, E., Soderlund, G., Ohlsson, I., Boiwe, T. and Brlnden, C. I. (1974) FEBS Let?. 44, 200-204 21 Webb, L. E., Hill, E. J. and Banaszak, L. J. (1973) Biochemistry 12, 5101-5109 22 Banaszak, L.J. and Bradshaw, R.A. (1975) in The Enzymes (Boyer, P. D., ed.), Vol XI, 3rd Edn, pp. 369-396, Academic Press, New York 23 Wimmer, M. J. and Harrison, J. H. (1975) J. Eiol. Chem. 250, 8768-8773 24 Moon, K., Piszkiewicz, D. and Smith, E. L. (1972) Proc. Nat. Acad. Sci. U.S.A. 69, 138&1383 25 Wootton, J. C., Chambers, G. K., Holder, A.A., Baron, A. J., Taylor, J. G., Fincham, J. R. S., Blumenthal, K: M., Moon, K. and Smith, E. L. (1974) Proc. Nat. Acad. Sci. U.S.A. 71, 43614365 26 Austen, B. M., Nyc, J. F., Degani, Y. and Smith, E. L. (1975) Proc. Nat. Acad. Sci. U.S.A. 72,48914894