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.