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

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. AULDCenter for Biochemical and Biophysical Sciences and Medicine and Department of Pathology, Harvard Medical School, and Brigham and Women's HospitalBoston, MA 02115

Contributed by Bert L. Vallee, October 10, 1989

ABSTRACT The x-ray crystallographic structures of 12zinc enzymes have been chosen as standards of reference toidentify the ligands to the catalytic and structural zinc atoms ofother members of their respective enzyme families. Univer-sally, H20 is a ligand and critical component of the catalyticallyactive zinc sites. In addition, three protein side chains bind tothe catalytic zinc atom, whereas four protein ligands bind to thestructural zinc atom. The geometry and coordination numberof zinc can vary greatly to accommodate particular ligands.Zinc forms complexes with nitrogen and oxygen just as readilyas with sulfur, and this is reflected in catalytic zinc sites havinga binding frequency of His >> Glu > Asp = Cys, three ofwhich bind to the metal atom. The systematic spacing betweenthe ligands is striking. For all catalytic zinc sites except thecoenzyme-dependent alcohol dehydrogenase, the first two lig-ands are separated by a "short spacer" consisting of 1 to 3amino acids. These ligands are separated from the third ligandby a "long spacer" of -20 to -120 amino acids. The shortspacer enables formation of a primary bidentate zinc complex,whereas the long spacer contributes flexibility to the coordi-nation sphere, which can poise the zinc for catalysis as well asbring other catalytic and substrate binding groups into appo-sition with the active site. The H20 is activated by ionization,polarization, or poised for displacement. Collectively, the dataimply that the preferred mechanistic pathway for activating thewater-e.g., zinc hydroxide or Lewis acid catalysis-will bedetermined by the identity of the other three ligands and theirspacing.

In the last three decades the biological role of zinc, like thatof a number of transition metals, has become most readilyapparent in enzymatic catalysis. Zinc is the only metal,however, that is essential in the function of at least oneenzyme in each one of the six classes established by theInternational Union of Biochemistry. Among these zincenzymes, the hydrolases are most abundant. Zinc enzymesoccur in all phyla, leaving no doubt regarding the essentialityof 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 zincenzymes has been accomplished by x-ray crystallographicanalysis. All other experimental approaches had proven to beunsatisfactory. Structures have now been obtained for 12zinc enzymes representing four of the six enzyme classes(Table 1). For these the details of coordination are nowthoroughly known, and their structures therefore representstandards of reference. We here examine the zinc ligands atthe active sites of these enzymes and compare them withthose in the sequences of other members of the same proteinfamily. The results should ultimately permit conclusionsregarding the conformations of the protein ligands that arerequired so that they can interact with zinc; these, in turn,

Table 1. Reported crystal structures of zinc enzymesClass Type

I OxidoreductaseAlcohol dehydrogenase

II TransferaseAspartate carbamoyltransferase

III HydrolaseCarboxypeptidase ACarboxypeptidase BDD carboxypeptidaseThermolysinBacillus cereus neutral protease,B-LactamaseAlkaline phosphatasePhospholipase C

IV LyaseCarbonic anhydrase ICarbonic anhydrase II

V IsomeraseNone

VI LigaseNone

may relate to the specificity of these enzymes and theirmechanisms of action.

MATERIALS AND METHODSComputer and literature searches have served to ascertainsequences, zinc content, and functional characteristics offamilies of enzymes corresponding to those of known struc-ture. A family of enzymes is here defined as a group ofproteins related by common ancestry as revealed by theirhomology and with identical or very similar functions. Boththe National Biomedical Research Foundation and Gen-Bank/Los Alamos data base files of the Molecular BiologyComputer Research Resource at Harvard Medical Schoolwere employed. The number of enzymes explicitly shown tocontain zinc by metal analysis and of others whose content isputative and inferred, based on their inhibition by metal-binding agents and/or activation with zinc, greatly exceedsthe number of enzymes whose three-dimensional structureshave been determined.

RESULTSIn the following, carboxypeptidases A and B of bovinepancreas, thermolysin, the neutral protease of Bacillus ther-moproteolyticus, the neutral protease of Bacillus cereus,carbonic anhydrases I and II of human erythro-tytes, and thedimeric alcohol dehydrogenase of horse liver serve as the

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

220

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Proc. Natl. Acad. Sci. USA 87 (1990) 221

Bovine A*

Rat Al

Rat A2

LG

TG

AG

Bovine B C G F

Crayfish B GG

RatB CGF

69 72

I SR I

SR V

IAR V~

F AAR V

I AR V

AR V

196

W I T

W V T

NV V T

W I S

N I A

N I S

F LS I

F I S I

F TL

Y LT I

Y LT F

Y LT 1|

SYSa

S Y S Q

SYSQ

SYSa

S Y S Q

SYSQ

FIG. 1. Zinc ligands of carboxypeptidases. Lightly shaded boxesdenote 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 ligandsbased on the structure of the standard of reference.

standards of reference for those members of their respectivefamilies of known sequence but unknown three-dimensionalstructure. Our specific objective here is to compare theidentities of the zinc ligands at their putative catalytic andstructural zinc-binding sites and the amino acid sequences intheir immediate vicinities. However, we stress some generalimplications of our findings that may pertain to the functionsof zinc and other metal active sites and, hence, the design ofenzymatically active model systems and the discernment ofthe mechanisms of such enzymes.

Class III: Hydrolases. Metalloexoproteinases. Carboxy-peptidase A (EC 3.4.17.1) has been considered a prototypefor all zinc proteases (1, 2). It contains 1 mol of zinc essentialfor activity per mol of Mr 34,600. X-ray structure analysis ofthe bovine A and B enzymes has revealed that zinc binds tothe same three protein ligands (L1-L3) in both enzymes-His-69, Glu-72, and His-196-and a H20 molecule (3, 4).His-69 (L1) and Glu-72 (L2) are separated by 2 amino acidresidues, henceforth referred to as the "short spacer," andGlu-72 and His-196 (L3) are separated by 123 amino acidresidues, henceforth referred to as the "long spacer." Theseresidues are completely conserved for six carboxypeptidaseA and B types from bovine, rat, and crayfish sources (Fig. 1).In addition, the specific amino acids in the vicinity of theseresidues are also 95% conserved.

Metalloendoproteinases. Thermolysin (EC 3.4.24.4) isrepresentative of a number of bacterial metalloproteinaseswith a pH optimum near neutrality which has defined them asneutral proteases. It contains 1 mol of zinc essential foractivity and 4 mol of calcium per mol of Mr 34,000 (5),presumably for protein stabilization. The x-ray crystal struc-ture of thermolysin (6) also reveals three protein ligands-His-142, His-146, and Glu-166-and a H20 molecule ligatedto 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.

In contrast to the carboxypeptidases, in this instance twohistidine residues, His-142 (L1) and His-146 (L2), are nearestneighbors, separated by a short spacer of 3 amino acids; the19 residue long spacer of thermolysin between His-146 (L2)and Glu-166 (L3) is significantly shorter than that of carbox-ypeptidase. In all bacterial neutral proteases sequenced sofar, five of the eight residues bordering Glu-166 (L3) areidentical, and the other three are closely similar (Fig. 2). Thusthe short and long amino acid spacers are constant andcharacteristic for each metalloprotease family. However,while the metal ligands for both families are identical, 2histidine and 1 glutamic acid, their order in the sequence (His,Glu, His versus His, His, Glu) is not; the other details pointedout also differ distinctly. This contrasts with the mechanisticsimilarities that have been emphasized; the potential signif-icance of these structural identities to those of functionrequires further exploration.t

Class IV: Lyases. X-ray crystal structures have been re-ported for-two of the three forms of carbonic anhydrase (EC4.2.1.1)-I (9) and 11 (10)-present in human erythrocytes.

In this case three histidines bind the zinc ligands, and againa H20 molecule fills the fourth coordination- site. A singleamino acid short spacer separates L1 (His-94) from L2(His-96); the seven amino acids surrounding these ligands in15 different carbonic anhydrases are 95% similar (Fig. 3). A22-amino acid, long spacer arm supplies L3 (His-119). Four ofthe eight amino acids surrounding it are identical for 15carbonic anhydrases sequenced, and the remaining fouramino acids show a high degree of similarity.

Class I: Oxidoreductases. Horse liver alcohol dehydroge-nase (EC 1.1.1.1) is a NAD(H)-dependent dimeric enzymecontaining two zinc atoms per monomer. It represents theonly zinc enzyme examined by x-ray crystallography (11) sofar in which the active-site zinc ligands differ somewhat fromall others studied. The catalytic zinc (Fig. 4) is bound to onehistidine and two cysteine residues; Cys-46 (L1) is separatedfrom His-67 (L2) by a 19-amino acid segment, constituting theshort spacer. This is the only relatively long nearest-neighborshort spacer distance of L1 and L2 in any one of these zincenzymes. Again, a water molecule is the fourth ligand. Thesequences about both Cys-46 and His-67 are again similar,but residue 47 has undergone a number of genetic mutations.

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

B. thermoproteolyticus

B. cereus

B. stearothermophilus

B. subtilis

B. amyloliquefaciens

142 146

VVA ELT AVT

V I G ELT AVT

V VG ELT AVT

V TA EMT GVT

VTA EMT GVT

166

-G A IN A I SD

_

G ALN

G A I N

G A LN

G A LN|

- ........ ....

A I SD

A MSD

S

S

F S D

FSD

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

Biochemistry: Vallee and Auld

222 Biochemistry: Vallee and Auld

119.................................... --

S A E LS G E LS A E LS A E LS S E L

A A E LA A E LA A ELA A EL ;D A E LA A E LA A E LA A E L

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 ofNAD(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 coenzyme-dependent zinc enzymes.We emphasize these particular details in the alcohol de-

hydrogenase structure, as it is the only one so far in whichboth zinc and a coenzyme are required for activity. Conceiv-ably, the need to accommodate this circumstance might beresponsible, in part, for the ligand design at an active sitewhich differs from that of all the other zinc enzymes, both as

regards the cysteine ligands and the length of the spacerbetween L1 and L2.A long spacer segment of 106 amino acids separates His-67

(L2) from Cys-174 (L3). Four of the eight amino acidssurrounding this cysteine are invariant, whereas the otherfour are very similar. The homology around this cysteineresidue should be noted, considering the broad evolutionaryrange of these alcohol dehydrogenase sequences.The second zinc atom of dimeric alcohol dehydrogenases,

bound to four cysteines, is not directly involved in enzymaticactivity. Its ligands, Cys-97, -100, -103, and -111, are sepa-rated by only 2, 2, and 7 residues, respectively (Fig. 5). Theintervening amino acid sequences now vary considerablyamong the family of alcohol dehydrogenase enzymes incontrast to the highly invariant nature observed for theresidues in the vicinity of the catalytic zinc ligands (Fig. 4).

Horse E

Human CX

Human 6

Human Y

Human TEHuman XRat X

MouseRat

*

Maize 1*

Maize 2

Pea*

46

T G I R S D

V G I GT D

V G I R T D

A G I R S D

T S L H T D

T A V H T D

T A V H T D

T G V RS D

T GV R S D

T S L H T D

T A L H T D

T S L H T D

DISCUSSIONEarly efforts to account for selective binding of zinc atenzyme active sites rested heavily on geochemical knowl-edge. The distinctive predominance of zinc sulfides in zincores-e.g., sphalerite, wurtzite, and galena-enhanced theview that zinc much prefers sulfur ligands (13).The stability constants of zinc coordination complexes,

largely with mono- or bidentate ligands, did not mitigate thisperception (14). Multidentate zinc complex ions synthesizedsince then have confirmed that zinc forms complexes withnitrogen and oxygen just as readily as with sulfur ligands, as

reflected in zinc enzymes by imidazole, sulfhydryl, andcarboxyl groups of histidine, cysteine, and glutamic andaspartic acids, respectively (Table 2).For each catalytic zinc site x-ray crystallography identifies

the zinc ligands as a combination of three of these four typesof 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 mostcommon (Table 2). Thus, two histidine residues are charac-teristic for the hydrolases, carboxypeptidase A (3), carbox-ypeptidase B (4), thermolysin (6), B. cereus neutral protease(7), phospholipase C (15), and alkaline phosphatase (16),whereas three histidines are typical for the lyases, carbonicanhydrase 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

67l AG

I L G

L G

I LG

I V GI LG

I LG

V L GVL G

I F G

L GF G

E AA

E AA

E AA

E AA

E GA

E GA

E GA

E GA

E AG

E AGE AG

174

C L I G G F S T

CL I G G F S T

CL I G G F S T

CL I G G FST

C LL G G FST

CL L G G IST

CL L G G IST

C LI G G FST

C LI G G FST

CV L S G IST

CI L S G IST

CI L S G ICT

FIG. 4. Active-site zinc ligands for dimeric alcohol dehydrogenases. For key to figures, see Fig. 1.

94 96F....... _.._FF

F

FF

_

F T:

Human IBovine IMouse IHorseRabbitMonkey:Human WBovine 11

*

Mouse 11 *Rabbit 11 *Sheep 11Chicken 11Human IIIBovine III

*

Horse 111

OF F_-F Q FT Q FV Q FS Q FF Q FI Q FVQ FI OFQ F

VQ FV OFRQ FRQ FRQ F

W G SWG IW G NW G SW G KW G SW G S.

W G SW G S

W G SW G SW G SW G SW G S

F

F

F

F

L

L

V A H WL V H WL V H WL V H WL V H WI V H W

L V H WL V H WL V H WL \I H WL V H W

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

-

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

Proc. Natl. Acad. Sci. USA 87 (1990) 223

orse EHuman CX

Human OHuman Y

Human rLHuman X

Rat X

MouseRat *

Maize 1*

Maize 2Pea*

P QP QP QP LP QP QP QP QG EG EG E I

103

V K

I KV KI KF LF LF LI KI KH KH KH K

111

HP EGNF LKNP E S N Y L KNP E S N Y L KNP E S N Y L KSP L T N L G KNP K T N L Q KNP K T N L Q KHP E S N F S RHP E S N L C QSA E S N M D LSE E S N M D LSE 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 sitewith two cysteine zinc ligands (Cys-46 and Cys-174). Theneighbor of one of these (Cys-47) additionally accommodatesthe phosphate of NAD(H).

In four of these enzymes, L3 is glutamate and, in one ofthem, L3 is aspartate, consistent with the oxygen donors ofzinc complex ions. Yet, in enzymes overall, zinc prefers theimidazole nitrogen, in contrast with surmises based ongeochemistry (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 aspartatecarbamoyltransferase (19), thought by some to be structuralzinc sites. Four cysteines are involved in binding the zinc. Inboth instances, the cysteines are spaced closely together inthe linear sequence, the interval being 2, 2, and 7 for alcoholdehydrogenase and 4, 22, and 2 for aspartate carbamoyl-transferase.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 acidsseparate L1 and L2, the first two ligands, in 10 of the 11enzymes in Table 2. This suggests that the proximity of theL1 and L2 protein residues, when properly oriented, facili-tates the formation of a primary bidentate zinc complex. It isequally characteristic for an active zinc site that, in the linearsequence, a relatively long spacer of from =20 to -120residues separates L3 from either of the first two ligands. Thisthird protein ligand (Table 2) generally comes from theC-terminal side of L1 and L2. While adding stability to zinccoordination, such a long spacer arm could also responsiblyparticipate in the three-dimensional alignment of the activesite, bringing other catalytic and substrate-binding groupsinto 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 thecatalytic process calls for a suitable alignment of amino acidresidues that can provide for both metal chelation andcoenzyme binding sites. Remarkably, this has been accom-plished in alcohol dehydrogenase by (i) using residues 46 and47 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 -3to 20 amino acids. The active zinc site of alcohol dehydro-genase is also the only 1 among the 11 zinc enzymes here citedas structural standards that comprises only one histidineresidue (Fig. 4).The long spacers that participate in the formation of the

catalytic sites imply that the zinc coordination geometryresulting from its interaction with the putative bidentate zinc

complex is much more flexible than that of structural sites,where interligand distances are much shorter. Closely spacedligands at structural zinc sites could be consistent with a rolefor zinc in stabilizing both protein overall structure and localconformation, analogous perhaps to disulfide bonds and theresults of interaction of calcium with some proteins. On theone hand, such an arrangement could likely impart rigidity tothat region of the molecule in which the interacting ligandsoccur. On the other hand, the long spacings of the active sitealso contribute flexibility to coordination numbers and ge-ometries that might poise the zinc for catalysis and create an

entatic state, while allowing the changes that take place whensubstrates and/or products interact during catalysis. More-over, they could be instrumental in bringing about productiveconformations by suitably aligning and organizing thoseadditional amino acid side chains that participate in thecatalytic process, including substrate binding and involve-ment of an outer-sphere ligand, the coordination of whichcould activate water. Equally important, flexible coordina-tion would provide the potential for conformational changesand generate a substrate-binding pocket. Variations in spacerlength may, therefore, affect differences in substrate speci-ficity, the functions of water, and the details of catalyticmechanisms.The characteristically short (1-3 amino acid) and long

(=20-120 amino acid) spacers that the catalytic zinc sitesshare could also help decipher zinc sites of as yet undefinedrole-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 metallo-

Table 2. Zinc ligands and their spacing for the catalytic zincEnzyme L1 X L2 Y L3 L4

Carbonic anhydrase I His 1 His 22 His (C) H20Carbonic anhydrase II His 1 His 22 His (C) H20,3-Lactamase His 1 His 121 His (C) H20DD-Carboxypeptidase His 2 His 40 His (N) H20Thermolysin His 3 His 19 Glu (C) H20B. cereus neutralprotease His 3 His 19 Glu (C) H20

Carboxypeptidase A His 2 Glu 123 His (C) H20Carboxypeptidase B His 2 Glu 123 His (C). H20Phospholipase C His 3 Glu 13 His (N) H20Alkaline phosphatase Asp 3 His 80 His (C) H20Alcohol dehydrogenase Cys 20 His 106 Cys (C) H20X 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. L3comes from either the amino (N) or the carboxyl (C) portion of theprotein.

Biochemistry: Vallee and Auld

224 Biochemistry: Vallee and Auld

HO

ZnZ-O

IONIZATION

H20

ZnI'l -\ I

-B--H--OH

Zn'I-,

POLARIZATION

S

Zn

DISPLACEMENT

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

proteins seem to represent a variation on the present themeto be detailed elsewhere.J

In all catalytically active zinc sites, H20 is the fourth ligand(L4) (Table 2) and a critical component. Ultimately, this watermolecule is activated by ionization, polarization, or poisedfor displacement once within the zinc coordination sphere(Fig. 6). On the one hand, ionization of the activated water orits polarization by a base form ofan active-site amino acid canprovide hydroxide ions at neutral pH; on the other hand,ready displacement of the water can lead to Lewis acidcatalysis by the catalytic zinc. Collectively, the results implythat the preferred mechanistic pathway for activating thewater will be determined by the identity of the other threeligands and their spacing. This is assisted, of course, by otheractive-site residues, the nature of which then determines thedetailed mechanisms of the catalytic reactions.These structural features of metalloenzymes reemphasize

the importance of protein folding and conformation known tounderlie the generation of functional molecules. In zincenzymes, they may be expressed, in part, by the seeminginstructions that the long spacer arm contains to createsuitable zinc coordination numbers and geometries. Metal-dependent systems may thereby gain new attention for prob-ing the folding process.The factors highlighted here also bear on the design of

enzyme model systems. We consider the catalytic potential ofzinc enzymes to depend on the characteristics ofthe short andlong spacers and the environment that they create for the metalligands. One might expect that, minimally, models would have

tThe structures of a number of copper and iron proteins also seemto conform to the spacer format. Thus, the iron-sulfur clustercomplex of aconitase is coordinated to Cys-359, Cys-422, andCys-425 (21). Furthermore, in the ascorbate oxidase from zucchini(22), a single copper is bound to His-446, Cys-508, His-513, andMet-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. Inthese cases, too, the details of amino acid spacing may provide askeleton for the interaction with the catalytic metal ion that deservesinspection.

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

This work was supported by grants from the Endowment forResearch in Human Biology, Inc. (Boston).

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