Proc. Natl. Acad. Sci. USA Vol. 87, pp. 220-224, January 1990 Biochemistry

Active-site zinc ligands and activated H20 of zinc enzymes (amino acid sequence/metalloenzymes/metalloproteins/structure-function/x-ray crystallography)

BERT L. VALLEE* AND DAVID S. AULD Center for Biochemical and Biophysical Sciences and Medicine and Department of Pathology, Harvard Medical School, and Brigham and Women's Hospital Boston, MA 02115

Contributed by Bert L. Vallee, October 10, 1989

The x-ray crystallographic structures of 12 ABSTRACT zinc enzymes have been chosen as standards of reference to identify the ligands to the catalytic and structural zinc atoms of other members of their respective enzyme families. Universally, H20 is a ligand and critical component of the catalytically active zinc sites. In addition, three protein side chains bind to the catalytic zinc atom, whereas four protein ligands bind to the structural zinc atom. The geometry and coordination number of zinc can vary greatly to accommodate particular ligands. Zinc forms complexes with nitrogen and oxygen just as readily as with sulfur, and this is reflected in catalytic zinc sites having a binding frequency of His >> Glu > Asp = Cys, three of which bind to the metal atom. The systematic spacing between the ligands is striking. For all catalytic zinc sites except the coenzyme-dependent alcohol dehydrogenase, the first two ligands are separated by a "short spacer" consisting of 1 to 3 amino acids. These ligands are separated from the third ligand by a "long spacer" of -20 to -120 amino acids. The short spacer enables formation of a primary bidentate zinc complex, whereas the long spacer contributes flexibility to the coordination sphere, which can poise the zinc for catalysis as well as bring other catalytic and substrate binding groups into apposition with the active site. The H20 is activated by ionization, polarization, or poised for displacement. Collectively, the data imply that the preferred mechanistic pathway for activating the water-e.g., zinc hydroxide or Lewis acid catalysis-will be determined by the identity of the other three ligands and their spacing.

Table 1. Reported crystal structures of zinc enzymes Class Type I Oxidoreductase Alcohol dehydrogenase II Transferase Aspartate carbamoyltransferase III Hydrolase Carboxypeptidase A Carboxypeptidase B DD carboxypeptidase Thermolysin Bacillus cereus neutral protease

,B-Lactamase IV V

VI

Alkaline phosphatase Phospholipase C Lyase Carbonic anhydrase I Carbonic anhydrase II Isomerase None Ligase None

may relate to the specificity of these enzymes and their mechanisms of action.

MATERIALS AND METHODS Computer and literature searches have served to ascertain sequences, zinc content, and functional characteristics of families of enzymes corresponding to those of known structure. A family of enzymes is here defined as a group of proteins related by common ancestry as revealed by their homology and with identical or very similar functions. Both the National Biomedical Research Foundation and GenBank/Los Alamos data base files of the Molecular Biology Computer Research Resource at Harvard Medical School were employed. The number of enzymes explicitly shown to contain zinc by metal analysis and of others whose content is putative and inferred, based on their inhibition by metalbinding agents and/or activation with zinc, greatly exceeds the number of enzymes whose three-dimensional structures have been determined.

In the last three decades the biological role of zinc, like that of a number of transition metals, has become most readily apparent in enzymatic catalysis. Zinc is the only metal, however, that is essential in the function of at least one enzyme in each one of the six classes established by the International Union of Biochemistry. Among these zinc enzymes, the hydrolases are most abundant. Zinc enzymes occur in all phyla, leaving no doubt regarding the essentiality of this element to all forms of life. Unambiguous identification of zinc ligands and their modes of coordination both at the active and structural sites of zinc enzymes has been accomplished by x-ray crystallographic analysis. All other experimental approaches had proven to be unsatisfactory. Structures have now been obtained for 12 zinc enzymes representing four of the six enzyme classes (Table 1). For these the details of coordination are now thoroughly known, and their structures therefore represent standards of reference. We here examine the zinc ligands at the active sites of these enzymes and compare them with those in the sequences of other members of the same protein family. The results should ultimately permit conclusions regarding the conformations of the protein ligands that are required so that they can interact with zinc; these, in turn,

RESULTS In the following, carboxypeptidases A and B of bovine pancreas, thermolysin, the neutral protease of Bacillus thermoproteolyticus, the neutral protease of Bacillus cereus, carbonic anhydrases I and II of human erythro-tytes, and the dimeric alcohol dehydrogenase of horse liver serve as the

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviation: L1, L2, L3, and L4, the first, second, third, and fourth zinc-binding ligand, respectively. *To whom reprint requests should be addressed.

220

Biochemistry: Vallee and Auld 69

Bovine A Rat Al

*

LG I

72

Proc. Natl. Acad. Sci. USA 87 (1990)

In contrast to the carboxypeptidases, in this instance two histidine residues, His-142 (L1) and His-146 (L2), are nearest neighbors, separated by a short spacer of 3 amino acids; the 19 residue long spacer of thermolysin between His-146 (L2) and Glu-166 (L3) is significantly shorter than that of carboxypeptidase. In all bacterial neutral proteases sequenced so far, five of the eight residues bordering Glu-166 (L3) are identical, and the other three are closely similar (Fig. 2). Thus the short and long amino acid spacers are constant and characteristic for each metalloprotease family. However, while the metal ligands for both families are identical, 2 histidine and 1 glutamic acid, their order in the sequence (His, Glu, His versus His, His, Glu) is not; the other details pointed out also differ distinctly. This contrasts with the mechanistic similarities that have been emphasized; the potential significance of these structural identities to those of function requires further exploration.t Class IV: Lyases. X-ray crystal structures have been reported for- two of the three forms of carbonic anhydrase (EC 4.2.1.1)-I (9) and 11 (10)-present in human erythrocytes. In this case three histidines bind the zinc ligands, and again a H20 molecule fills the fourth coordination- site. A single amino acid short spacer separates L1 (His-94) from L2 (His-96); the seven amino acids surrounding these ligands in 15 different carbonic anhydrases are 95% similar (Fig. 3). A 22-amino acid, long spacer arm supplies L3 (His-119). Four of the eight amino acids surrounding it are identical for 15 carbonic anhydrases sequenced, and the remaining four amino acids show a high degree of similarity. Class I: Oxidoreductases. Horse liver alcohol dehydrogenase (EC 1.1.1.1) is a NAD(H)-dependent dimeric enzyme containing two zinc atoms per monomer. It represents the only zinc enzyme examined by x-ray crystallography (11) so far in which the active-site zinc ligands differ somewhat from all others studied. The catalytic zinc (Fig. 4) is bound to one histidine and two cysteine residues; Cys-46 (L1) is separated from His-67 (L2) by a 19-amino acid segment, constituting the short spacer. This is the only relatively long nearest-neighbor short spacer distance of L1 and L2 in any one of these zinc enzymes. Again, a water molecule is the fourth ligand. The sequences about both Cys-46 and His-67 are again similar, but residue 47 has undergone a number of genetic mutations.

196

SR IW I T F LS I

SYSa

Rat A2

SR VW V T F I S I S Y S Q AG IAR V~NV V T F TL SYSQ

Bovine B

C G FF

Crayfish B

GG I AR VN I A

Y LT F S Y S Q

RatB

CGF

Y LT 1| SYSQ

TG

AAR

VW I S Y LT I

AR VN I S

221

SYSa

FIG. 1. Zinc ligands of carboxypeptidases. Lightly shaded boxes denote the enzyme(s) x-ray standard of reference for each family. Asterisks denote those for which zinc was not measured directly. Black vertical columns indicate the proposed metal binding ligands based on the structure of the standard of reference.

standards of reference for those members of their respective families of known sequence but unknown three-dimensional structure. Our specific objective here is to compare the identities of the zinc ligands at their putative catalytic and structural zinc-binding sites and the amino acid sequences in their immediate vicinities. However, we stress some general implications of our findings that may pertain to the functions of zinc and other metal active sites and, hence, the design of enzymatically active model systems and the discernment of the mechanisms of such enzymes. Class III: Hydrolases. Metalloexoproteinases. Carboxypeptidase A (EC 3.4.17.1) has been considered a prototype for all zinc proteases (1, 2). It contains 1 mol of zinc essential for activity per mol of Mr 34,600. X-ray structure analysis of the bovine A and B enzymes has revealed that zinc binds to the same three protein ligands (L1-L3) in both enzymesHis-69, Glu-72, and His-196-and a H20 molecule (3, 4). His-69 (L1) and Glu-72 (L2) are separated by 2 amino acid residues, henceforth referred to as the "short spacer," and Glu-72 and His-196 (L3) are separated by 123 amino acid residues, henceforth referred to as the "long spacer." These residues are completely conserved for six carboxypeptidase A and B types from bovine, rat, and crayfish sources (Fig. 1). In addition, the specific amino acids in the vicinity of these residues are also 95% conserved. Metalloendoproteinases. Thermolysin (EC 3.4.24.4) is representative of a number of bacterial metalloproteinases with a pH optimum near neutrality which has defined them as neutral proteases. It contains 1 mol of zinc essential for activity and 4 mol of calcium per mol of Mr 34,000 (5), presumably for protein stabilization. The x-ray crystal structure of thermolysin (6) also reveals three protein ligandsHis-142, His-146, and Glu-166-and a H20 molecule ligated to the zinc (Fig. 2). The B. cereus neutral protease is 73% homologous with thermolysin, and its x-ray crystal structure (7) shows near identity to that of thermolysin.

tA large number of so-called metalloendoproteases have now been recognized in virtually all phyla, isolated and/or cloned, sequenced, and characterized partially or completely, and they are zinc enzymes (8). The structure of thermolysin has played a key role in efforts to identify their zinc-binding ligands, kinetics, and mechanism. In many of these enzymes, 2 histidines are found, separated by 3 amino acids resembling the short spacer of thermolysin. However, a third glutamic acid ligand to zinc, which the thermolysin structure would predict to be -20 amino acids away, has not been found there or in its vicinity. Apparently, the location of the third zinc ligand cannot be specified in the absence of either an x-ray or NMR structure or of exhaustive mutagenic data.

142

146

166

B. thermoproteolyticus

VVA

ELT AVT

B. cereus

VIG

ELT

AVT

G ALN

A I SD

B. stearothermophilus

V VG

ELT AVT

G A I N

A MSD

B. subtilis

V TA

EMT

GVT

G A LN

S FSD

B. amyloliquefaciens

VTA

EMT

GVT

G A LN| S FSD

-G A IN A I SD _

-

FIG. 2. Zinc ligands of thermolysins. For key to figures, see Fig. 1.

........ ....

222

..

Proc. Natl. Acad. Sci. USA 87 (1990)

Biochemistry: Vallee and Auld

96

94

OF _-F .......F_.._ F F Q F F T Q F V Q F F S Q F F T: F Q F FF I Q F F VQ F

Human I Bovine I Mouse I Horse Rabbit Monkey :Human W Bovine 11 * Mouse 11 * Rabbit 11 * Sheep 11 Chicken 11 Human III Bovine III * Horse 111

_

OF

F

Q F

F

VQ F V OF RQ F RQ F RQ F

F

I

L

L

W G S WG I W G N W G S W G K W G S W G S.

W W W W W W W

G G G G G G G

119 --

.............

S S S S S S S

L L L L L

A G A A S

E E E E E

A A A A D A A A

A A A A A A A A

L L E L L E L L EL EL ; L E L L E L L E L E L- L

FIG. 3. Zinc ligands of carbonic anhydrases. For key to figures,

*

*

Maize 2 Pea*

V

V A T T T T T T T T

G I G I G I S L A V A V G V GV S L A L S L

see

AG

RS D

l

GT D

I L G L G I LG I V G I LG I LG V L G VL G I F G L G F G

H T D H T D H T D

RS R S H T H T H T

D D D D D

\I H W V H W

V H W V/ H W V H W V H W

Fig. 1.

174

67

R T D R S D

HW VV H W V H W

Early efforts to account for selective binding of zinc at enzyme active sites rested heavily on geochemical knowledge. The distinctive predominance of zinc sulfides in zinc ores-e.g., sphalerite, wurtzite, and galena-enhanced the view that zinc much prefers sulfur ligands (13). The stability constants of zinc coordination complexes, largely with mono- or bidentate ligands, did not mitigate this perception (14). Multidentate zinc complex ions synthesized since then have confirmed that zinc forms complexes with nitrogen and oxygen just as readily as with sulfur ligands, as reflected in zinc enzymes by imidazole, sulfhydryl, and carboxyl groups of histidine, cysteine, and glutamic and aspartic acids, respectively (Table 2). For each catalytic zinc site x-ray crystallography identifies the zinc ligands as a combination of three of these four types of residues (Figs. 1-4). H20 is the universal ligand at all of the catalytic zinc sites, but considering the side chains, histidine is by far the most common (Table 2). Thus, two histidine residues are characteristic for the hydrolases, carboxypeptidase A (3), carboxypeptidase B (4), thermolysin (6), B. cereus neutral protease (7), phospholipase C (15), and alkaline phosphatase (16), whereas three histidines are typical for the lyases, carbonic anhydrase I and II (9, 10), and the hydrolases B-lactamase (17) and DD-carboxypeptidase of Streptomyces albus G (18). The only catalytic zinc site with only one histidine is that of

46 T G I

L V H W L V H W I V H W

DISCUSSION

It is one of the two sites for binding the phosphate of NAD(H). Variations of this residue are found among the a, ,3, y, ir, or X human alcohol dehydrogenase isozymes (12). Such considerations may prove to pertain to other coenzymedependent zinc enzymes. We emphasize these particular details in the alcohol dehydrogenase structure, as it is the only one so far in which both zinc and a coenzyme are required for activity. Conceivably, the need to accommodate this circumstance might be responsible, in part, for the ligand design at an active site which differs from that of all the other zinc enzymes, both as regards the cysteine ligands and the length of the spacer between L1 and L2. A long spacer segment of 106 amino acids separates His-67 (L2) from Cys-174 (L3). Four of the eight amino acids surrounding this cysteine are invariant, whereas the other four are very similar. The homology around this cysteine residue should be noted, considering the broad evolutionary range of these alcohol dehydrogenase sequences. The second zinc atom of dimeric alcohol dehydrogenases, bound to four cysteines, is not directly involved in enzymatic activity. Its ligands, Cys-97, -100, -103, and -111, are separated by only 2, 2, and 7 residues, respectively (Fig. 5). The intervening amino acid sequences now vary considerably among the family of alcohol dehydrogenase enzymes in contrast to the highly invariant nature observed for the residues in the vicinity of the catalytic zinc ligands (Fig. 4).

Horse E Human CX Human 6 Human Y Human TE Human X X Rat Mouse Rat Maize 1

V A H W L V H W L V H W

S S S S S

E AA E

AA

E

AA

E

AA

E

GA

E

GA

E

GA

E

GA

E AG E AG E AG

C L I G

G F S T

CL I CL I CL I C LL CL L CL L C LI C LI CV L CI L

G

G F S T

G

G F S T

G G

G G G G

G

G G G

FST FST IST IST

G FST

G FST

S

G IST

S

G IST G ICT

CI L S

FIG. 4. Active-site zinc ligands for dimeric alcohol dehydrogenases. For key to figures,

see

Fig. 1.

Biochemistry: Vallee and Auld

Proc. Natl. Acad. Sci. USA 87 (1990) 103

orse

E

Human CX

Human O Human Human rL Human X Rat X Mouse Rat * Maize 1 Maize 2 Pea* Y

*

P Q P P

Q Q

P L P Q P Q P Q P Q G E G E G E

I

111

V

K

HP

I

K K K L L L K K

NP NP NP SP NP NP HP HP SA SE SE

V

I F F F I I H H H

K K K

EGNF S N Y S N Y

E E E L K K E E E E

S T T T S S S S

N N N N N N N N E S N

Y L L L F L M M

M

LK L K L K L K G K Q K Q K S R C Q D L D L D L

FIG. 5. Structural-site zinc ligands for dimeric alcohol dehydrogenases. For key to figures,

alcohol dehydrogenase (11); it is further the only active site with two cysteine zinc ligands (Cys-46 and Cys-174). The neighbor of one of these (Cys-47) additionally accommodates the phosphate of NAD(H). In four of these enzymes, L3 is glutamate and, in one of them, L3 is aspartate, consistent with the oxygen donors of zinc complex ions. Yet, in enzymes overall, zinc prefers the imidazole nitrogen, in contrast with surmises based on geochemistry (see above). On the other hand, cysteines are the sole ligands of the second zinc atom ofthe dimeric alcohol dehydrogenases (Fig. 5) and of the zinc atom of the regulatory subunit of aspartate carbamoyltransferase (19), thought by some to be structural zinc sites. Four cysteines are involved in binding the zinc. In both instances, the cysteines are spaced closely together in the linear sequence, the interval being 2, 2, and 7 for alcohol dehydrogenase and 4, 22, and 2 for aspartate carbamoyltransferase. The systematic spacing between the ligands to the catalytic zinc atom is striking, and its importance cannot be ignored (20). Short spacers consisting of only 1, 2, or 3 amino acids separate L1 and L2, the first two ligands, in 10 of the 11 enzymes in Table 2. This suggests that the proximity of the L1 and L2 protein residues, when properly oriented, facilitates the formation of a primary bidentate zinc complex. It is equally characteristic for an active zinc site that, in the linear sequence, a relatively long spacer of from =20 to -120 residues separates L3 from either of the first two ligands. This third protein ligand (Table 2) generally comes from the C-terminal side of L1 and L2. While adding stability to zinc coordination, such a long spacer arm could also responsibly participate in the three-dimensional alignment of the active site, bringing other catalytic and substrate-binding groups into apposition. Alchohol dehydrogenase is the only coenzyme-dependent zinc enzyme whose three-dimensional structure is known. The conjoint involvement of both zinc and NADH in the catalytic process calls for a suitable alignment of amino acid residues that can provide for both metal chelation and coenzyme binding sites. Remarkably, this has been accomplished in alcohol dehydrogenase by (i) using residues 46 and 47 as zinc and NADH binding ligands, respectively, (ii) providing two cysteines as ligands to the active site zinc, and (iii) elongating the short spacer between L1 and L2 from -3 to 20 amino acids. The active zinc site of alcohol dehydrogenase is also the only 1 among the 11 zinc enzymes here cited as structural standards that comprises only one histidine residue (Fig. 4). The long spacers that participate in the formation of the catalytic sites imply that the zinc coordination geometry resulting from its interaction with the putative bidentate zinc

223

see

Fig. 1.

complex is much more flexible than that of structural sites, where interligand distances are much shorter. Closely spaced ligands at structural zinc sites could be consistent with a role for zinc in stabilizing both protein overall structure and local conformation, analogous perhaps to disulfide bonds and the results of interaction of calcium with some proteins. On the one hand, such an arrangement could likely impart rigidity to that region of the molecule in which the interacting ligands occur. On the other hand, the long spacings of the active site also contribute flexibility to coordination numbers and geometries that might poise the zinc for catalysis and create an entatic state, while allowing the changes that take place when substrates and/or products interact during catalysis. Moreover, they could be instrumental in bringing about productive conformations by suitably aligning and organizing those additional amino acid side chains that participate in the catalytic process, including substrate binding and involvement of an outer-sphere ligand, the coordination of which could activate water. Equally important, flexible coordination would provide the potential for conformational changes and generate a substrate-binding pocket. Variations in spacer length may, therefore, affect differences in substrate specificity, the functions of water, and the details of catalytic mechanisms. The characteristically short (1-3 amino acid) and long (=20-120 amino acid) spacers that the catalytic zinc sites share could also help decipher zinc sites of as yet undefined role-e.g., in phospholipase C (Asp-55, His-69, His-118, Asp-122) (15) and alkaline phosphatase (Asp-51, Asp-369, His-370) (16). These sites and those seen in other metalloTable 2. Zinc ligands and their spacing for the catalytic zinc Y L1 X L2 Enzyme L3 L4 Carbonic anhydrase I His 1 His 22 His (C) H20 Carbonic anhydrase II His 1 His 22 His (C) H20 His 1 His 121 His (C) H20 ,3-Lactamase 40 His (N) H20 DD-Carboxypeptidase His 2 His His 3 His 19 Glu (C) H20 Thermolysin B. cereus neutral His 3 His 19 Glu (C) H20 protease His 2 Glu 123 His (C) H20 Carboxypeptidase A His 2 Glu 123 His (C). H20 Carboxypeptidase B His 3 Glu 13 His (N) H20 Phospholipase C Alkaline phosphatase 80 His (C) H20 Asp 3 His Alcohol dehydrogenase Cys 20 His 106 Cys (C) H20 X is the number of amino acids between L1 and L2; Y is the number of amino acids between L3 and its nearest zinc ligand neighbor. L3 comes from either the amino (N) or the carboxyl (C) portion of the protein.

Biochemistry: Vallee and Auld

224

Proc. Natl. Acad. Sci. USA 87 (1990)

-B--H--OH

HO H20

Zn 'I-,

Zn

Z-O

Zn I'l

IONIZATION

-\

IPOLARIZATION

S

Zn DISPLACEMENT

FIG. 6. Schematic of the functions of the H20 ligand in active zinc sites of zinc enzymes. S, substrate; B, base.

proteins

seem to

to be detailed

represent

a

variation on the present theme

elsewhere.J

In all catalytically active zinc sites, H20 is the fourth ligand (L4) (Table 2) and a critical component. Ultimately, this water molecule is activated by ionization, polarization, or poised for displacement once within the zinc coordination sphere (Fig. 6). On the one hand, ionization of the activated water or its polarization by a base form of an active-site amino acid can provide hydroxide ions at neutral pH; on the other hand, ready displacement of the water can lead to Lewis acid catalysis by the catalytic zinc. Collectively, the results imply that the preferred mechanistic pathway for activating the water will be determined by the identity of the other three ligands and their spacing. This is assisted, of course, by other active-site residues, the nature of which then determines the detailed mechanisms of the catalytic reactions. These structural features of metalloenzymes reemphasize the importance of protein folding and conformation known to underlie the generation of functional molecules. In zinc enzymes, they may be expressed, in part, by the seeming instructions that the long spacer arm contains to create suitable zinc coordination numbers and geometries. Metaldependent systems may thereby gain new attention for probing the folding process. The factors highlighted here also bear on the design of enzyme model systems. We consider the catalytic potential of zinc enzymes to depend on the characteristics of the short and long spacers and the environment that they create for the metal ligands. One might expect that, minimally, models would have structures of a number of copper and iron proteins also seem to conform to the spacer format. Thus, the iron-sulfur cluster complex of aconitase is coordinated to Cys-359, Cys-422, and

tThe

Cys-425 (21). Furthermore, in the ascorbate oxidase from zucchini (22), a single copper is bound to His-446, Cys-508, His-513, and Met-518, and a trinuclear copper cluster involves the ligands His-62, His-64, His-106, His-108, His-449, His-451, His-507, and His-509. In these cases, too, the details of amino acid spacing may provide a skeleton for the interaction with the catalytic metal ion that deserves inspection.

to mimic these features to achieve the potentials of catalysis and specificity, analogous to those of zinc enzymes.

This work was supported by grants from the Endowment for Research in Human Biology, Inc. (Boston). 1. Vallee, B. L., Galdes, A., Auld, D. S. & Riordan, J. F. (1983) in Zinc Enzymes, ed. Spiro, T. G. (Wiley, New York), pp. 26-75. 2. Auld, D. S. & Vallee, B. L. (1987) in Hydrolytic Enzymes, eds. Neuberger, A. & Brocklehurst, K. (Elsevier, New York), pp. 201-255. 3. Quiocho, F. A. & Lipscomb, W. N. (1971) Adv. Prot. Chem. 25, 1-58. 4. Schmid, M. F. & Herriott, J. R. (1976) J. Mol. Biol. 103, 175-190. 5. Holmquist, B. & Vallee, B. L. (1974) J. Biol. Chem. 249, 4601-4607. 6. Matthews, B. W., Jansonius, J. N., Colman, P. M., Schoenborn, B. P. & Dupourque, D. (1972) Nature New Biol. (London) 238, 37-41. 7. Pauptit, R. A., Karlsson, R., Picot, D., Jenkins, J. A., NiklausReimer, A.-S. & Jansonius, J. N. (1988) J. Mol. Biol. 168,

525-537. 8. Vallee, B. L. & Auld, D. S., in Proceedings of the Matrix Metalloproteinase Conference, eds. Birkedal-Hanson, H., Werb, Z., Welgus, H. & Van Wart, H. (Sandestin, FL), in press. 9. Kannan, K. K., Notstrand, B., Fridborg, K., Logren, S., Orlsson, A. & Petef, M. (1975) Proc. Nati. Acad. Sci. USA 72, 51-55. 10. Liljas, A., Kannan, K. K., Bergsten, P. C., Waara, I., Fridborg, K., Strandberg, B., Carlbom, V., Jarup, L., Logren, S. & Petef, M. (1972) Nature (London) 235, 131-137. 11. Branden, C. I., Jornvall, M., Eklund, M. & Furugren, B. (1975) in Enzymes, ed. Boyer, P. D. (Academic, New York), 3rd Ed., Vol. 11, p. 103. 12. Jornvall, H., Hoog, J.-O., von Bahr-Lindstrom, H. & Vallee, B. L. (1987) Biochemistry 84, 2580-2584. 13. Matthewson, C. H. (1959) Zinc (Penfield, New York). 14. Bjerrum, J., Schwarzenbach, G. & Sillen, L. G. (1957-1958) Spec. Publ. Chem. Soc. 17. 15. Hough, E., Hansen, L. K., Birknes, B., Jynge, K., Hansen, S., Horvik, A., Little, C., Dodson, E. & Derewenda, Z. (1989) Nature (London) 338, 357-360. 16. Wyckoff, H. W., Handschumacher, M., Krishna Murthy, H. M., & Sowadski, J. M. (1983) in Adv. Enzymol. Relat. Areas Mol. Biol. 55, 453-480. 17. Sutton, B. J., Artymiuk, P. J., Cordero-Borboa, A. E., Little, C., Phillips, D. C. & Waley, S. G. (1987) Biochem. J. 248, 181-188. 18. Dideberg, O., Charlier, P., Dive, G., Joris, B., Frere, J. M. & Ghuysen, J. M. (1982) Nature (London) 299, 469-470. 19. Honazatko, R. B., Crawford, J. L., Monaco, H. L., Ladner, J. E., Edwards, B. F. P., Evans, D. R., Warren, S. G., Wiley, D. C., Ladner, R. C. & Lipscomb, W. N. (1982) J. Mol. Biol. 160, 219-263. 20. Vallee, B. L. & Auld, D. S. (1980) FEBS Lett. 257, 138-140. 21. Robbins, A. M. & Stout, C. D. (1989) Proteins Struct. Funct. Genet. 5, 289-312. 22. Messerschmidt, A., Rossi, A., Lodenstein, R., Huber, R., Bolognesi, M., Gatti, G., Marchesini, A., Petruzzelli, R. & Finazzi-Agro, A. (1989) J. Mol. Biol. 203, 513-529.