Active-Site Zinc Ligands and Activated H20 of Zinc Enzymes (Amino Acid Sequence/Metalloenzymes/Metalloproteins/Structure-Function/X-Ray Crystallography) BERT L

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

ABSTRACT The x-ray crystallographic structures of 12 Table 1. Reported crystal structures of zinc enzymes zinc enzymes have been chosen as standards of reference to Class Type identify the ligands to the catalytic and structural zinc atoms of other members of their respective enzyme families. Univer- I Oxidoreductase sally, H20 is a ligand and critical component ofthe catalytically Alcohol dehydrogenase active zinc sites. In addition, three protein side chains bind to II Transferase the catalytic zinc atom, whereas four protein ligands bind to the Aspartate carbamoyltransferase structural zinc atom. The geometry and coordination number III Hydrolase of zinc can vary greatly to accommodate particular ligands. Carboxypeptidase A Zinc forms complexes with nitrogen and oxygen just as readily Carboxypeptidase B as with sulfur, and this is reflected in catalytic zinc sites having DD carboxypeptidase > = Thermolysin a binding frequency of His >> Glu Asp Cys, three of Bacillus cereus neutral protease which bind to the metal atom. The systematic spacing between ,B-Lactamase the ligands is striking. For all catalytic zinc sites except the Alkaline phosphatase coenzyme-dependent alcohol dehydrogenase, the first two lig- Phospholipase C ands are separated by a "short spacer" consisting of 1 to 3 IV Lyase amino acids. These ligands are separated from the third ligand Carbonic anhydrase I by a "long spacer" of -20 to -120 amino acids. The short Carbonic anhydrase II spacer enables formation of a primary bidentate zinc complex, V Isomerase whereas the long spacer contributes flexibility to the coordi- None nation sphere, which can poise the zinc for catalysis as well as VI Ligase bring other catalytic and substrate binding groups into appo- None sition with the active site. The H20 is activated by ionization, polarization, or poised for displacement. Collectively, the data may relate to the specificity of these enzymes and their imply that the preferred mechanistic pathway for activating the of action. water-e.g., zinc hydroxide or Lewis acid catalysis-will be mechanisms determined by the identity of the other three ligands and their spacing. MATERIALS AND METHODS Computer and literature searches have served to ascertain In the last three decades the biological role of zinc, like that sequences, zinc content, and functional characteristics of of a number of transition metals, has become most readily families of enzymes corresponding to those of known struc- apparent in enzymatic catalysis. Zinc is the only metal, ture. A family of enzymes is here defined as a group of however, that is essential in the function of at least one their enzyme in each one of the six classes established by the proteins related by common ancestry as revealed by International Union of Biochemistry. Among these zinc homology and with identical or very similar functions. Both enzymes, the hydrolases are most abundant. Zinc enzymes the National Biomedical Research Foundation and Gen- occur in all phyla, leaving no doubt regarding the essentiality Bank/Los Alamos data base files of the Molecular Biology of this element to all forms of life. Computer Research Resource at Harvard Medical School Unambiguous identification ofzinc ligands and their modes were employed. The number of enzymes explicitly shown to of coordination both at the active and structural sites of zinc contain zinc by metal analysis and ofothers whose content is enzymes has been accomplished by x-ray crystallographic putative and inferred, based on their inhibition by metal- analysis. All other experimental approaches had proven to be binding agents and/or activation with zinc, greatly exceeds unsatisfactory. Structures have now been obtained for 12 the number of enzymes whose three-dimensional structures zinc enzymes representing four of the six enzyme classes have been determined. (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 RESULTS the active sites of these enzymes and compare them with In the following, carboxypeptidases A and B of bovine those in the sequences of other members of the same protein pancreas, thermolysin, the neutral protease of Bacillus ther- family. The results should ultimately permit conclusions moproteolyticus, the neutral protease of Bacillus cereus, regarding the conformations of the protein ligands that are carbonic anhydrases I and II of human erythro-tytes, and the required so that they can interact with zinc; these, in turn, dimeric alcohol dehydrogenase of horse liver serve as the

The publication costs of this article were defrayed in part by page charge Abbreviation: L1, L2, L3, and L4, the first, second, third, and fourth payment. This article must therefore be hereby marked "advertisement" zinc-binding ligand, respectively. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 220 Downloaded by guest on October 2, 2021 Biochemistry: Vallee and Auld Proc. Natl. Acad. Sci. USA 87 (1990) 221 69 72 196 In contrast to the carboxypeptidases, in this instance two histidine residues, His-142 (L1) and His-146 (L2), are nearest Bovine A LG I SR IW I T F LS I SYSa neighbors, separated by a short spacer of 3 amino acids; the * Rat Al TG SR VW V T F I S I S Y S Q 19 residue long spacer of thermolysin between His-146 (L2) and Glu-166 (L3) is significantly shorter than that of carbox- Rat A2 AG IAR V~NV V T F TL SYSQ ypeptidase. In all bacterial neutral proteases sequenced so far, five of the eight residues bordering Glu-166 (L3) are Bovine B C G FF AAR VW I S Y LT I SYSa identical, and the other three are closely similar (Fig. 2). Thus the short and long amino acid spacers are constant and Crayfish B GG I AR VN I A Y LT F S Y S Q characteristic for each metalloprotease family. However, RatB CGF AR VN I S Y LT 1| SYSQ while the metal ligands for both families are identical, 2 histidine and 1 glutamic acid, their order in the sequence (His, FIG. 1. Zinc ligands of carboxypeptidases. Lightly shaded boxes Glu, His versus His, His, Glu) is not; the other details pointed denote the enzyme(s) x-ray standard of reference for each family. out also differ distinctly. This contrasts with the mechanistic Asterisks denote those for which zinc was not measured directly. similarities that have been emphasized; the potential signif- Black vertical columns indicate the proposed metal binding ligands icance of these structural identities to those of function based on the structure of the standard of reference. requires further exploration.t Class IV: Lyases. X-ray crystal structures have been re- standards of reference for those members of their respective ported for-two of the three forms of carbonic anhydrase (EC families of known sequence but unknown three-dimensional 4.2.1.1)-I (9) and 11 (10)-present in human erythrocytes. structure. Our specific objective here is to compare the In this case three histidines bind the zinc ligands, and again identities of the zinc ligands at their putative catalytic and a H20 molecule fills the fourth coordination- site. A single structural zinc-binding sites and the amino acid sequences in amino acid short spacer separates L1 (His-94) from L2 their immediate vicinities. However, we stress some general (His-96); the seven amino acids surrounding these ligands in implications of our findings that may pertain to the functions 15 different carbonic anhydrases are 95% similar (Fig. 3). A of zinc and other metal active sites and, hence, the design of 22-amino acid, long spacer arm supplies L3 (His-119). Four of enzymatically active model systems and the discernment of the eight amino acids surrounding it are identical for 15 the mechanisms of such enzymes. carbonic anhydrases sequenced, and the remaining four Class III: Hydrolases. Metalloexoproteinases. Carboxy- amino acids show a high degree of similarity. peptidase A (EC 3.4.17.1) has been considered a prototype Class I: Oxidoreductases. Horse liver alcohol dehydroge- for all zinc proteases (1, 2). It contains 1 mol of zinc essential nase (EC 1.1.1.1) is a NAD(H)-dependent dimeric enzyme for activity per mol of Mr 34,600. X-ray structure analysis of containing two zinc atoms per monomer. It represents the the bovine A and B enzymes has revealed that zinc binds to only zinc enzyme examined by x-ray crystallography (11) so the same three protein ligands (L1-L3) in both enzymes- far in which the active-site zinc ligands differ somewhat from His-69, Glu-72, and His-196-and a H20 molecule (3, 4). all others studied. The catalytic zinc (Fig. 4) is bound to one His-69 (L1) and Glu-72 (L2) are separated by 2 amino acid histidine and two cysteine residues; Cys-46 (L1) is separated residues, henceforth referred to as the "short spacer," and from His-67 (L2) by a 19-amino acid segment, constituting the Glu-72 and His-196 (L3) are separated by 123 amino acid short spacer. This is the only relatively long nearest-neighbor residues, henceforth referred to as the "long spacer." These short spacer distance of L1 and L2 in any one of these zinc residues are completely conserved for six carboxypeptidase enzymes. Again, a water molecule is the fourth ligand. The A and B types from bovine, rat, and crayfish sources (Fig. 1). sequences about both Cys-46 and His-67 are again similar, In addition, the specific amino acids in the vicinity of these but residue 47 has undergone a number ofgenetic mutations. residues are also 95% conserved. Metalloendoproteinases. Thermolysin (EC 3.4.24.4) is tA large number of so-called metalloendoproteases have now been representative of a number of bacterial metalloproteinases recognized in virtually all phyla, isolated and/or cloned, sequenced, with a pH optimum near neutrality which has defined them as and characterized partially or completely, and they are zinc en- neutral proteases. It contains 1 mol of zinc essential for zymes (8). The structure of thermolysin has played a key role in activity and 4 mol of calcium per mol of Mr 34,000 (5), efforts to identify their zinc-binding ligands, kinetics, and mecha- presumably for protein stabilization. The x-ray crystal struc- nism. In many of these enzymes, 2 histidines are found, separated ture of thermolysin (6) also reveals three protein ligands- by 3 amino acids resembling the short spacer of thermolysin. However, a third glutamic acid ligand to zinc, which the thermolysin His-142, His-146, and Glu-166-and a H20 molecule ligated structure would predict to be -20 amino acids away, has not been to the zinc (Fig. 2). The B. cereus neutral protease is 73% found there or in its vicinity. Apparently, the location of the third homologous with thermolysin, and its x-ray crystal structure zinc ligand cannot be specified in the absence of either an x-ray or (7) shows near identity to that of thermolysin. NMR structure or of exhaustive mutagenic data. 142 146 166 B. thermoproteolyticus VVA ELT AVT -G A IN A I SD

_ - ...... B. cereus V I G 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 F S D B. amyloliquefaciens VTA EMT GVT G A LN| S FSD FIG. 2. Zinc ligands of thermolysins. For key to figures, see Fig. 1. Downloaded by guest on October 2, 2021 222 Biochemistry: Vallee and Auld Proc. Natl. Acad. Sci. USA 87 (1990) 94 96 119 I F F W S V A H W Human OF _- ...... _.._ G ...... -- Bovine I F Q F F WG I S.... A E L L V H W F F W G N S...... G E L L V H W Mouse I T Q Horse V Q F _ W G S S A E L L V H W Rabbit S Q F F W G K S A E L L V H W Monkey F Q F F T: W G S S S E L I V H W FF :Human W I Q F W G S. Bovine 11 VQ F F A A E L L VV H WW * W G S F H W Mouse 11 * I OF W G S A A E L L V Rabbit 11 * Q F F A A EL L \I H W Sheep 11 VQ F F W G S A A EL ; L V H W Chicken 11 V OF W G S D A E L V H W Human III RQ F W G S A A E L L V/ H W Bovine III RQ F L W G S A A E L L V H W * L A A E L L V H W Horse 111 RQ F W G S -

FIG. 3. Zinc ligands of carbonic anhydrases. For key to figures, see Fig. 1.

It is one of the two sites for binding the phosphate of DISCUSSION NAD(H). Variations of this residue are found among the a, Early efforts to account for selective binding of zinc at ,3, y, ir, or X human alcohol dehydrogenase isozymes (12). enzyme active sites rested heavily on geochemical knowl- Such considerations may prove to pertain to other coenzyme- edge. The distinctive predominance of zinc sulfides in zinc dependent zinc enzymes. ores-e.g., sphalerite, wurtzite, and galena-enhanced the We emphasize these particular details in the alcohol de- view that zinc much prefers sulfur ligands (13). hydrogenase structure, as it is the only one so far in which The stability constants of zinc coordination complexes, both zinc and a coenzyme are required for activity. Conceiv- largely with mono- or bidentate ligands, did not mitigate this ably, the need to accommodate this circumstance might be perception (14). Multidentate zinc complex ions synthesized responsible, in part, for the ligand design at an active site since then have confirmed that zinc forms complexes with which differs from that of all the other zinc enzymes, both as nitrogen and oxygen just as readily as with sulfur ligands, as regards the cysteine ligands and the length of the spacer reflected in zinc enzymes by imidazole, sulfhydryl, and between L1 and L2. carboxyl groups of histidine, cysteine, and glutamic and A long spacer segment of 106 amino acids separates His-67 aspartic acids, respectively (Table 2). (L2) from Cys-174 (L3). Four of the eight amino acids For each catalytic zinc site x-ray crystallography identifies surrounding this cysteine are invariant, whereas the other the zinc ligands as a combination of three of these four types four are very similar. The homology around this cysteine of residues (Figs. 1-4). residue should be noted, considering the broad evolutionary H20 is the universal ligand at all of the catalytic zinc sites, range of these alcohol dehydrogenase sequences. but considering the side chains, histidine is by far the most The second zinc atom of dimeric alcohol dehydrogenases, common (Table 2). Thus, two histidine residues are charac- bound to four cysteines, is not directly involved in enzymatic teristic for the hydrolases, carboxypeptidase A (3), carbox- activity. Its ligands, Cys-97, -100, -103, and -111, are sepa- ypeptidase B (4), thermolysin (6), B. cereus neutral protease rated by only 2, 2, and 7 residues, respectively (Fig. 5). The (7), phospholipase C (15), and alkaline phosphatase (16), intervening amino acid sequences now vary considerably whereas three histidines are typical for the lyases, carbonic among the family of alcohol dehydrogenase enzymes in anhydrase I and II (9, 10), and the hydrolases B-lactamase contrast to the highly invariant nature observed for the (17) and DD-carboxypeptidase of Streptomyces albus G (18). residues in the vicinity of the catalytic zinc ligands (Fig. 4). The only catalytic zinc site with only one histidine is that of

46 67 174 Horse E T G I R S D l AG E AA C L I G G F S T

Human CX V G I GT D I L G CL I G G F S T Human 6 V G I R T D L G E AA CL I G G F S T Human Y A G I R S D I LG E AA CL I G G FST Human TE T S L H T D I V G E AA C LL G G FST E Human X T A V H T D I LG GA CL L G G IST Rat X T A V H T D I LG E GA CL L G G IST Mouse T G V RS D V L G E GA C LI G G FST Rat T GV R S D VL G E GA C LI G G FST * Maize 1 T S L H T D I F G E AG CV L S G IST * Maize 2 T A L H T D L G E AG CI L S G IST

Pea* T S L H T D F G E AG CI L S G ICT

FIG. 4. Active-site zinc ligands for dimeric alcohol dehydrogenases. For key to figures, see Fig. 1. Downloaded by guest on October 2, 2021 Biochemistry: Vallee and Auld Proc. Natl. Acad. Sci. USA 87 (1990) 223

103 111

orse E V K HP EGNF LK Human CX P Q I K NP E S N Y L K Human O P Q V K NP E S N Y L K Human Y P Q I K NP E S N Y L K Human rL P L F L SP L T N L G K Human X P Q F L NP K T N L Q K Rat X P Q F L NP K T N L Q K Mouse P Q I K HP E S N F S R

Rat * P Q I K HP E S N L C Q Maize 1 G E H K SA E S N M D L Maize 2 * G E H K SE E S N M D L Pea* G E I H K SE E S N M D L

FIG. 5. Structural-site zinc ligands for dimeric alcohol dehydrogenases. For key to figures, see Fig. 1.

alcohol dehydrogenase (11); it is further the only active site complex is much more flexible than that of structural sites, with two cysteine zinc ligands (Cys-46 and Cys-174). The where interligand distances are much shorter. Closely spaced neighbor of one ofthese (Cys-47) additionally accommodates ligands at structural zinc sites could be consistent with a role the phosphate of NAD(H). for zinc in stabilizing both protein overall structure and local In four of these enzymes, L3 is glutamate and, in one of conformation, analogous perhaps to disulfide bonds and the them, L3 is aspartate, consistent with the oxygen donors of results of interaction of calcium with some proteins. On the zinc complex ions. Yet, in enzymes overall, zinc prefers the one hand, such an arrangement could likely impart rigidity to imidazole nitrogen, in contrast with surmises based on that region of the molecule in which the interacting ligands geochemistry (see above). occur. On the other hand, the long spacings of the active site On the other hand, cysteines are the sole ligands of the also contribute flexibility to coordination numbers and ge- second zinc atom ofthe dimeric alcohol dehydrogenases (Fig. ometries that might poise the zinc for catalysis and create an 5) and of the zinc atom of the regulatory subunit of aspartate entatic state, while allowing the changes that take place when carbamoyltransferase (19), thought by some to be structural substrates and/or products interact during catalysis. More- zinc sites. Four cysteines are involved in binding the zinc. In over, they could be instrumental in bringing about productive both instances, the cysteines are spaced closely together in conformations by suitably aligning and organizing those the linear sequence, the interval being 2, 2, and 7 for alcohol additional amino acid side chains that participate in the dehydrogenase and 4, 22, and 2 for aspartate carbamoyl- transferase. catalytic process, including substrate binding and involve- The systematic spacing between the ligands to the catalytic ment of an outer-sphere ligand, the coordination of which zinc atom is striking, and its importance cannot be ignored could activate water. Equally important, flexible coordina- (20). Short spacers consisting of only 1, 2, or 3 amino acids tion would provide the potential for conformational changes separate L1 and L2, the first two ligands, in 10 of the 11 and generate a substrate-binding pocket. Variations in spacer enzymes in Table 2. This suggests that the proximity of the length may, therefore, affect differences in substrate speci- L1 and L2 protein residues, when properly oriented, facili- ficity, the functions of water, and the details of catalytic tates the formation of a primary bidentate zinc complex. It is mechanisms. equally characteristic for an active zinc site that, in the linear The characteristically short (1-3 amino acid) and long sequence, a relatively long spacer of from =20 to -120 (=20-120 amino acid) spacers that the catalytic zinc sites residues separates L3 from either ofthe first two ligands. This share could also help decipher zinc sites of as yet undefined third protein ligand (Table 2) generally comes from the role-e.g., in phospholipase C (Asp-55, His-69, His-118, C-terminal side of L1 and L2. While adding stability to zinc Asp-122) (15) and alkaline phosphatase (Asp-51, Asp-369, coordination, such a long spacer arm could also responsibly His-370) (16). These sites and those seen in other metallo- participate in the three-dimensional alignment of the active site, bringing other catalytic and substrate-binding groups Table 2. Zinc ligands and their spacing for the catalytic zinc into apposition. Alchohol dehydrogenase is the only coenzyme-dependent Enzyme L1 X L2 Y L3 L4 zinc enzyme whose three-dimensional structure is known. Carbonic anhydrase I His 1 His 22 His (C) H20 The conjoint involvement of both zinc and NADH in the Carbonic anhydrase II His 1 His 22 His (C) H20 catalytic process calls for a suitable alignment of amino acid ,3-Lactamase His 1 His 121 His (C) H20 residues that can provide for both metal chelation and DD-Carboxypeptidase His 2 His 40 His (N) H20 coenzyme binding sites. Remarkably, this has been accom- Thermolysin His 3 His 19 Glu (C) H20 plished in alcohol dehydrogenase by (i) using residues 46 and B. cereus neutral 47 as zinc and NADH binding ligands, respectively, (ii) protease His 3 His 19 Glu (C) H20 providing two cysteines as ligands to the active site zinc, and Carboxypeptidase A His 2 Glu 123 His (C) H20 (iii) elongating the short spacer between L1 and L2 from -3 Carboxypeptidase B His 2 Glu 123 His (C). H20 to 20 amino acids. The active zinc site of alcohol dehydro- Phospholipase C His 3 Glu 13 His (N) H20 genase is also the only 1 among the 11 zinc enzymes here cited Alkaline phosphatase Asp 3 His 80 His (C) H20 as structural standards that comprises only one histidine Alcohol dehydrogenase Cys 20 His 106 Cys (C) H20 residue (Fig. 4). X is the number ofamino acids between L1 and L2; Yis the number The long spacers that participate in the formation of the of amino acids between L3 and its nearest zinc ligand neighbor. L3 catalytic sites imply that the zinc coordination geometry comes from either the amino (N) or the carboxyl (C) portion of the resulting from its interaction with the putative bidentate zinc protein. Downloaded by guest on October 2, 2021 224 Biochemistry: Vallee and Auld Proc. Natl. Acad. Sci. USA 87 (1990) HO -B--H--OH to mimic these features to achieve the potentials of catalysis H20 and specificity, analogous to those of zinc enzymes. Zn Zn 'I-, This work was supported by grants from the Endowment for Z-O Zn Research in Human Biology, Inc. (Boston). IONIZATION I'l -\ IPOLARIZATION 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. S Neuberger, A. & Brocklehurst, K. (Elsevier, New York), pp. 201-255. Zn 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, DISPLACEMENT 175-190. 5. Holmquist, B. & Vallee, B. L. (1974) J. Biol. Chem. 249, FIG. 6. Schematic of the functions of the H20 ligand in active 4601-4607. zinc sites of zinc enzymes. S, substrate; B, base. 6. Matthews, B. W., Jansonius, J. N., Colman, P. M., Schoen- born, B. P. & Dupourque, D. (1972) Nature New Biol. (Lon- proteins seem to represent a variation on the present theme don) 238, 37-41. to be detailed elsewhere.J 7. Pauptit, R. A., Karlsson, R., Picot, D., Jenkins, J. A., Niklaus- In all catalytically active zinc sites, H20 is the fourth ligand Reimer, A.-S. & Jansonius, J. N. (1988) J. Mol. Biol. 168, (L4) (Table 2) and a critical component. Ultimately, this water 525-537. molecule is activated by ionization, polarization, or poised 8. Vallee, B. L. & Auld, D. S., in Proceedings of the Matrix for displacement once within the zinc coordination sphere Metalloproteinase Conference, eds. Birkedal-Hanson, H., Werb, Z., Welgus, H. & Van Wart, H. (Sandestin, FL), in (Fig. 6). On the one hand, ionization ofthe activated water or press. its polarization by a base form of an active-site amino acid can 9. Kannan, K. K., Notstrand, B., Fridborg, K., Logren, S., provide hydroxide ions at neutral pH; on the other hand, Orlsson, A. & Petef, M. (1975) Proc. Nati. Acad. Sci. USA 72, ready displacement of the water can lead to Lewis acid 51-55. catalysis by the catalytic zinc. Collectively, the results imply 10. Liljas, A., Kannan, K. K., Bergsten, P. C., Waara, I., Frid- that the preferred mechanistic pathway for activating the borg, K., Strandberg, B., Carlbom, V., Jarup, L., Logren, S. water will be determined by the identity of the other three & Petef, M. (1972) Nature (London) 235, 131-137. ligands and their spacing. This is assisted, of course, by other 11. Branden, C. I., Jornvall, M., Eklund, M. & Furugren, B. (1975) active-site the nature of which then determines the in Enzymes, ed. Boyer, P. D. (Academic, New York), 3rd Ed., residues, Vol. 11, p. 103. detailed mechanisms of the catalytic reactions. 12. Jornvall, H., Hoog, J.-O., von Bahr-Lindstrom, H. & Vallee, These structural features of metalloenzymes reemphasize B. L. (1987) Biochemistry 84, 2580-2584. the importance of protein folding and conformation known to 13. Matthewson, C. H. (1959) Zinc (Penfield, New York). underlie the generation of functional molecules. In zinc 14. Bjerrum, J., Schwarzenbach, G. & Sillen, L. G. (1957-1958) enzymes, they may be expressed, in part, by the seeming Spec. Publ. Chem. Soc. 17. instructions that the long spacer arm contains to create 15. Hough, E., Hansen, L. K., Birknes, B., Jynge, K., Hansen, S., suitable zinc coordination numbers and geometries. Metal- Horvik, A., Little, C., Dodson, E. & Derewenda, Z. (1989) dependent systems may thereby gain new attention for prob- Nature (London) 338, 357-360. the folding process. 16. Wyckoff, H. W., Handschumacher, M., Krishna Murthy, ing H. M., & Sowadski, J. M. (1983) in Adv. Enzymol. Relat. Areas The factors highlighted here also bear on the design of Mol. Biol. 55, 453-480. enzyme model systems. We consider the catalytic potential of 17. Sutton, B. J., Artymiuk, P. J., Cordero-Borboa, A. E., Little, zinc enzymes to depend on the characteristics ofthe short and C., Phillips, D. C. & Waley, S. G. (1987) Biochem. J. 248, long spacers and the environment that they create forthe metal 181-188. ligands. One might expect that, minimally, models would have 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, tThe structures of a number of copper and iron proteins also seem B. F. D. to conform to the spacer format. Thus, the iron-sulfur cluster J. E., Edwards, P., Evans, R., Warren, S. G., Wiley, complex of aconitase is coordinated to Cys-359, Cys-422, and D. C., Ladner, R. C. & Lipscomb, W. N. (1982) J. Mol. Biol. Cys-425 (21). Furthermore, in the ascorbate oxidase from zucchini 160, 219-263. (22), a single copper is bound to His-446, Cys-508, His-513, and 20. Vallee, B. L. & Auld, D. S. (1980) FEBS Lett. 257, 138-140. Met-518, and a trinuclear copper cluster involves the ligands His-62, 21. Robbins, A. M. & Stout, C. D. (1989) Proteins Struct. Funct. His-64, His-106, His-108, His-449, His-451, His-507, and His-509. In Genet. 5, 289-312. these cases, too, the details of amino acid spacing may provide a 22. Messerschmidt, A., Rossi, A., Lodenstein, R., Huber, R., skeleton for the interaction with the catalytic metal ion that deserves Bolognesi, M., Gatti, G., Marchesini, A., Petruzzelli, R. & inspection. Finazzi-Agro, A. (1989) J. Mol. Biol. 203, 513-529. Downloaded by guest on October 2, 2021