Proc. Natl Acad. Sci. USAVol. 78, No. 9, pp. 5455-5459, September 1981Biochemistry

Binding of ligands to the active site of carboxypeptidase A(enzyme activity/proteases/protein crystallography/solid-state activity of enzymes/enzyme inhibition kinetics)

D. C. REES AND W. N. LIPscoMB*Gibbs Chemical Laboratory, Harvard University, Cambridge, Massachusetts 02138

Contributed by William N. Lipscomb, June 1, 1981

ABSTRACT We compare the detailed binding modes of the39-amino acid inhibitor from potatoes, glycyl-L-tyrosine, the esteranalogue CH30C6H4(CO)CH2CH(CO0)C6H5, and indole acetateto the exopeptidase carboxypeptidase A (EC 3.4.17.1). In the po-tato inhibitor, cleavage of the COOH-terminal glycine-39 leavesa new carboxylate anion ofvaline-38 having one oxygen on zinc andthe other as a receptor of a hydrogen bond from tyrosine-248 ofcarboxypeptidase. Tyrosine-248 also receives a hydrogen bondfrom the amide proton of the originally penultimate peptide bondbetween tyrosine-37 and valine-38. This hydrogen bond suggestsproduct stabilization which is available to peptides and depsipep-tides but not to esters lacking an equivalent peptide bond (non-specific esters). Also, this structure may represent the interme-diate binding step for the uncleaved substrate as it moves alongthe binding subsites. In particular, this may be the binding modefor the substrate after association of the COOH-terminal regionof the substrate with the residues at binding subsite S2 (tyrosine-198, phenylalanine-279, and arginine-71) and preceding entry intothe catalytic site S;. These stabilized complexes allow some un-derstanding of the effect of indole acetate, shown here to bind inthe pocket at S' , as a competitive inhibitor for esters (for whichentry into S' precedes the rate-determining catalytic step for hy-drolysis) and as a noncompetitive inhibitor for peptides (for whichentry into S' is rate limiting). These results, including the bindingmode of the ester analogue, are consistent with the original pro-posal from x-ray studies that both esters and peptides are cleavedwith the carboxy terminus at S';, although not necessarily by thesame chemical steps.

A complete description ofthe mechanism ofan enzyme requiresunderstanding ofthe detailed succession ofbinding and catalyticsteps. The nature of the interactions between the enzyme andsubstrate as the substrate progresses through the bindinggroove and into the active site pocket may profoundly influencethe observed binding and catalytic characteristics. The mech-anism of the exopeptidase carboxypeptidase A (CPase A; pep-tidyl-L-amino-acid hydrolase, EC 3.4.17.1) has been discussedfrom this viewpoint (1). In this paper, we describe crystallo-graphic studies of the binding of a ketonic substrate, competi-tive and non-competitive inhibitors, and product molecules toCPase A in relation to the progression of binding steps and themechanisms of hydrolysis of peptides and esters.A significant result for the mechanistic interpretation of

CPase A inhibition studies is that noncompetitive and compet-itive inhibitors can bind to the same site on the enzyme. Thisobservation is inconsistent with an interpretation of inhibitionkinetics based on a simple Michaelis-Menten model in whichnoncompetitive and competitive inhibitors bind to distinct sites(2). For CPase A, the action of various inhibitors on peptidaseand esterase activity is more consistent with a multistep mech-

anism in which the kinetic effects ofan inhibitor depend on thedetails of the rate-determining step for a particular substrate.

MATERIALS AND METHODS

CPase A. (Cox), glycyl-L-tyrosine (Gly-Tyr), and indole-3-aceticacid (IAA) were purchased from Sigma and used without fur-ther purification. (-)-2-Benzyl-3-p-methoxybenzoylpropionate(BMBP) was a generous gift of E. T. Kaiser. Crystals of CPaseA were prepared and crosslinked as described (3). It must bestressed that the native CPase A crystals used for all crystallo-&raphic work (space group P21, unit cell parameters a = 51.60A, b = 60.27 A, c = 47.25 A, / = 97.270, with the crystal habitelongated along the a axis) exhibit one-third the activity of theenzyme in solution (4). These crystals are different from crystalsof CPase Ar (Anson) (space group P21, unit cell parameters a=50.9 A, b = 57.9 A, c = 45.0 A, 8 = 94.670, with the crystalhabit elongated along the b axis), which only exhibit 1/300thofthe activity ofthe enzyme in solution (5). The less-active formof the CPase A crystals generally has been used for biochemicalstudies (6). The distinction between these two forms must berecognized when comparisons of solution/crystal enzyme ac-tivities are discussed.

Crystal soaking conditions for the difference map studies ofGly-Tyr (7) and BMBP (3) binding to CPase A have been doc-umented. Crystals ofthe CPase A-IAA complex were preparedby soaking crosslinked crystals ofnative CPase A in 10mM IAA/0.1 M NaCV20 mM Veronal, pH 7.5, for 48 hr. Crystallizationconditions and structure determination ofthe complex betweenCPase A and the 39-amino acid inhibitor from potatoes (PCI)have been described (8). Especially relevant for this discussion,the CPase A-PCI complex was crystallized from solution in atrigonal crystal form that is unrelated to the crystal form of thenative enzyme. Moreover, there are two molecules ofthis com-plex in different crystallographic environments. Thus, this crys-tal structure provides an independent view of the CPase Astructure and CPase A-ligand interactions to complement thework on the native crystals.

Data collection and structure determination of the nativeenzyme (9) and of the complexes of CPase A with Gly-Tyr (7),BMBP (3), and PCI (8) at 1.75-, 2.0-, 2.8-, and 2.5 A resolution,respectively, are described elsewhere. Coordinates for the na-tive enzyme and the CPase A-PCI complex have been refinedto crystallographic R factors of 0.174 and 0.196 at 1.5- and 2.5A resolution, respectively, by using the restrained least squaresrefinement algorithm of Hendrickson and Konnert (10) (un-published data; M. Lewis, personal communication). A 2.3 Aresolution data set was collected from a single crystal of theCPase A-IAA complex on a Syntex P21 diffractometer at 200C.

Abbreviations: CPase A, carboxypeptidase A; IAA, indole-3-acetic acid;BMBP, (-)-2-benzyl-3-p-methoxybenzoylpropionate; PCI, potato car-boxypeptidase A inhibitor; AAT, arsanilazotyrosine-248.* To whom reprint requests should be addressed.

The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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5456 Biochemistry: Rees and Lipscomb

Data processing and graphics procedures have been described(9).

Least squares estimates of the occupancies of the ligands inthese complexes were obtained by the usual refinement pro-cedures (11). An isotropic temperature factor of 12 A2 was as-sumed for all ligand atoms. The ligand coordinates, obtainedfrom the graphics program, were not refined. Populations of43%, 54%, and 28% were obtained for the Gly-Tyr, IAA, andBMBP complexes with CPase A, respectively.

RESULTSThe active site region of CPase A in the native enzyme is illus-trated in Fig. 1 for comparison with the CPase A-ligand com-plexes described below.

Binding ofGly-Tyr. The binding interactions ofGly-Tyr withCPase A (7, 9) are illustrated in Fig. 2. Gly-Tyr is a slowlycleaved substrate and a competitive inhibitor for more rapidlycleaved peptide substrates. The tyrosine ring of Gly-Tyr oc-cupies the S' binding pocket; the carboxylate group forms a saltlink to arginine-145, and the peptide bond carbonyl oxygenbinds to the zinc. Interestingly, the amino nitrogen of Gly-Tyris statistically distributed between a coordination site on thezinc and a salt bridge to the carboxylate group ofglutamate-270.In response to the binding of Gly-Tyr, several pronounced con-formational changes occur in CPase A: the most prominent isthe movement, by about 8 A, of the tyrosine-248 ring from thenative "up" position to the "down" position, where the hydroxylproton may interact with the carboxylate group of Gly-Tyr.Smaller but significant changes occur in the conformation ofarginine-145, isoleucine-247, and glutamate-270. In addition,several water molecules are displaced upon binding of Gly-Tyr.The entropic effects associated with the release of bond watermay play a significant role in the thermodynamics of ligandbinding and catalysis.

Binding of BMBP. CPase A has been shown to catalyze astereospecific exchange ofone of the methylene protons of theketonic substrate BMBP (12). The moderate rotational freedomaround the ketone group suggests that BMBP should resemblean ester more closely than a peptide substrate. The major fea-tures ofBMBP binding are similar to those of Gly-Tyr (3): thephenyl ring occupies the S', binding site, the carboxylate groupbinds to arginine-145, and the carbonyl oxygen is coordinatedto the zinc (Fig. 3). The p-methoxybenzoyl group binds neartyrosine-198. The methylene group, which undergoes the ster-

eospecific proton exchange, is located near glutamate-270.Binding of IAA. IAA is a noncompetitive inhibitor of pepti-

dase activity but a competitive inhibitor ofesterase activity (13).The indole ring of IAA occupies S'1, and the carboxylate groupbinds to arginine-145 (Fig. 4). Only one binding site for IAA wasobserved at the concentration used in this experiment. In viewof the differences in inhibitor activity of IAA and Gly-Tyr, it isimportant that, to within the limits imposed by the dissimilar-ities in molecular structures ofthe two ligands, IAA and Gly-Tyrbind to the same region of the enzyme.

Binding ofPCI. The previous binding studies have been con-cerned with small ligands which bind to CPase A primarily atthe S', and Zn binding sites. The complex of CPase A with the39-amino acid PCI molecule indicates the interaction of an ex-tended substrate to CPase A (Fig. 5). The S's, Si, S2, and S3 sitesare occupied by glycine-39, valine-38, tyrosine-37, and proline-36, respectively, in the CPase A-PCI complex (8). (In the fol-lowing discussion, residue numbers below 40 will refer to thePCI molecule.) Because glycine-39 is hydrolyzed from the re-mainder of the inhibitor and is trapped in the S', pocket, theCPase A-PCI structure represents a product complex.The two atoms formerly joined by the cleaved peptide bond,

the amino nitrogen of glycine-39 and the carbonyl carbon ofvaline-38, are separated by 3.3 A in this complex. The new car-boxylate anion of valine-38 (Si binding site) has one oxygen onthe zinc and the other as a receptor of a hydrogen bond fromtyrosine-248. Tyrosine-248 is located in the "down" confor-mation, similar to that seen in the previously described com-plexes. Tyrosine-248 also receives a hydrogen bond from theamide proton ofvaline-38. The carboxyl group ofglutamate-270is more than 4 A from the carboxyl group of valine-38, pre-cluding the occurrence here ofa stable anhydride species. How-ever, this does not rule out an anhydride intermediate duringpeptide bond hydrolysis.

The carbonyl oxygen of tyrosine-37 (S2 binding site) is hy-drogen bonded to arginine-71. In position S3, proline-36 bindsto CPase A near the aromatic groups of tyrosine-198 and phen-ylalanine-279. The presence of the proline, and an intramole-cular hydrogen bond in PCI between the carbonyl oxygen ofproline-36 and the indole nitrogen of tryptophan-28, creates abend in the PCI chain as it leaves the active site region ofCPaseA. Consequently, a fifth binding site (S4), which was proposedon the basis of kinetic studies (14), could not be identified inthis work.

Outside of the differences in the active site region expected

FIG. 1. Active site region of CPase A in the un-liganded state.

Proc. Nad Acad. Sci. USA 78 (1981)

Proc. Nad Acad. Sci. USA 78 (1981) 5457

FIG. 2. Active site region in the CPase A-Gly-Tyr complex. The Gly-Tyr molecule is indicated bythe darker bonds.

from earlier crystallographic studies, the structures of CPase Adetermined from the native and CPase A-PCI crystals are thesame to within the limits of the resolution and refinement al-gorithms. The main chain coordinates of the three crystallo-graphically independent CPase A molecules solved from nativeand CPase A-PCI crystals may be superimposed on one anotherwith root mean square deviations between 0.26 and 0.42 A.Because the lattice contacts are different in these two crystalforms, the influence of the crystal packing on the CPase A con-formation is small. Consequently, there would appear to be lit-tle reason to suspect significant differences between the mo-lecular structures of CPase A in solution and in crystals,although flexible side chains such as tryosine-248 are particu-larly responsive to environmental changes, including chemicalmodification and binding of substrates and inhibitors.

DISCUSSION

Mechanistic Considerations. The structures of the com-plexes of Gly-Tyr, BMBP, IAA, and PCI with CPase A are con-sistent with the proposal that the productive binding of bothesters and peptides places the carboxy-terminal side chain ofthe substrate in the S'1 pocket; the bond to be cleaved is locatedbetween the S'1 and S1 sites (1). An alternate hypothesis, thatesters and peptides are cleaved in different sites, was based onstriking differences in binding and kinetic characteristics be-tween ester and peptide substrates (13, 15). An essential as-sumption behind this alternate view was that competitive in-hibitors bind in the same site as a substrate but noncompetitiveinhibitors bind in different sites. The binding of Gly-Tyr andLAA described here show that such an assumption is invalid-bothligands bind in S'I, with the carboxylate group on arginine-145.

0-

4X4

a-

The present observations support instead a model in which pep-tides and esters are cleaved in the same site, consistent with amultistep reaction having different rate-controlling steps foresters and peptides (1, 16). A simple model, in which entry ofthe carboxy-terminal residue into the binding site pocket S'1precedes the rate-determining catalytic step for esters but is therate-determining step for peptides, is in accord with most ofthecrystallographic and kinetic studies ofCPase A (1). This differ-ential behavior of peptides and esters would be primarily dueto more facile distortion of ester compared to peptide bonds.

Aside from these similarities in binding, however, differentdetailed mechanisms for hydrolysis may apply to different sub-strates, even though the cleavage would occur between S'1 andS1 (1). Although the present crystallographic experiments can-not distinguish between the anhydride and general base mech-anisms, and do not even eliminate the zinc-hydroxyl mecha-nism, they do permit inferences concerning structural aspectsof possible hydrolytic intermediates (1). For example, we canregard the PCI structure as a complex with both product mol-ecules positioned for resynthesis of the peptide bond and thenconsider the implications of this structure for the reverse re-action, peptide bond hydrolysis. Assume, for discussion, thegeneral base pathway for peptide bond hydrolysis and assumethat the carboxylate oxygen ofvaline-38 bound to the zinc is thecarbonyl oxygen of the original peptide bond. One plausiblecatalytic sequence is as follows. The oxygen of the water mol-ecule promoted by glutamate-270 attacks the carbonyl carbonof the scissile peptide bond. Tyrosine-248 donates a proton tothe released amino nitrogen, leaving a phenolate anion. Theorientation of tyrosine-248 in the CPase A-PCI complex sug-gests that this residue is now properly positioned to accept aproton from the attacking water molecule. Thus, the same water

FIG. 3. Comparison of the binding of Gly-Tyr(solid lines) and BMBP (dashed lines) to CPase A.The conformations of Zn, arginine-145, and gluta-mate-270 in the CPase A-Gly-Tyr complex are in-dicated. The active site is viewed from the same di-rection as in Fig. 1.

Biochemistry: Rees and Lipscomb

5458 Biochemistry: Rees and Lipscomb

FIG. 4. Comparison of the binding of Gly-Tyr(solid lines) and IAA (dashed lines) to CPase A. Seethe legend to Fig. 3 for additional details.

molecule that attacks the peptide bond could also provide theproton to regenerate tyrosine-248, without requiring a secondwater molecule for this function. (One alternative, in which bothglutamate-270 and Zn promote the attack of one water, wouldyield a newly formed carboxylate anion which has the carbonyloxygen, originally on Zn, finally oriented toward tyrosine-248;proton transfer might then require additional steps.) The for-mation ofa hydrogen bond between tyrosine-248 and the amideproton of the originally penultimate peptide bond between ty-rosine-37 and valine-38 (Fig. 5) also suggests a product stabili-zation interaction for peptides and depsipeptides. This hydro-gen bond could not be formed for esters lacking an equivalentpeptide bond (nonspecific esters), however, and may be re-sponsible for some ofthe kinetic differences between these twoclasses of esters.

Another important mechanistic question concerns entry ofthe substrate into the active site pocket. The interaction of thevaline-38 carboxyl group with the zinc may represent a penul-timate mode of substrate binding, which follows association ofthe carboxy-terminal region of the substrate with residues at S2(arginine-71, tyrosine-198, and phenylalanine-279). This bind-ing stage would immediately precede entry of the carboxy-ter-minal residue into the S'1 pocket. Because glycine-39 is un-necessary for PCI binding to CPase A (17) it is possible that theCPase A-PCI structure actually represents a complex that istrapped in this intermediate binding stage and is unable to pro-ceed to the true catalytic transition state due to steric interac-tions between CPase A and the remainder ofthe PCI molecule.

Role of Tyrosine in Catalysis. In all of the difference mapstudies of ligand binding to CPase A in native CPase A crystals,strong negative electron density is observed at the original po-sition of tyrosine-248 and positive density is present for thedown orientation of this residue. This observation requires thatbinding of ligands to CPase A induces the conformationalchange in tyrosine-248 from the up to the down conformation.An earlier assignment, due to errors in the phases, of 15-25%oftyrosine-248 liganded to Zn in the native CPase A crystals hasbeen revised downward by our refinement at 1.5-A resolution.The occupancy ofa tyrosine ring placed in this putative positionrefines to a value of -4% ± 10% at 1.5 A resolution. This im-

plies that a tyrosine-248-Zn interaction occurs in very low per-centage, if at all, in the native enzyme. This result and the ac-tivity of the crystals used in the x-ray structure determinationstrongly suggest that tyrosine-248 is up in the unliganded en-zyme and that binding of substrates induces the conformationalchange to the down position.

Spectroscopic studies of arsanilazotyrosine-248 CPase A(AAT-CPase A), however, have been interpreted as suggestingthat a tyrosine-248-Zn interaction is an essential prerequisitefor catalysis (6, 18-22), in direct contradiction to the conclusionfrom the crystallographic studies. Although AAT may chelateto the Zn in unliganded AAT-CPase A, it seems likely that thisinteraction is a property of the arsanilazo modification. Extrap-olation of this behavior to the unmodified enzyme may be mis-leading. AAT is able to form a bidendate chelate to the zinc, incontrast to the unidendate coordination that unmodified tyro-sine-248 must form. In addition, the arsanilic acid group, withboth an aromatic ring and an anionic acid function, containsstructural features ofpreferred substrates for CPase A, possiblyfacilitating binding of this group to the enzyme. Both of theseeffects will promote enhanced binding of AAT to the. zinc, incomparison to unmodified tyrosine-248. The pK for theAAT-Zn interaction is 7.7, in the middle ofthe pH-activity pro-file for unmodified CPase A. Consequently, the pK for the un-modified tyrosine-248-Zn interaction, if it occurs at all, mustbe greater than 7.7, placing it in the range where it would notoccur to a significant extent over most ofthe pH range for CPaseA activity. In addition, the kinetics of the AAT-Zn interactionindicate that this association is slower than the rate ofrapid sub-strate hydrolysis (1, 4), again suggesting that the tyrosine-248-Zn ligand is not involved in the catalytic mechanism ofunmodified CPase A.On the basis of resonance Raman studies of AAT-CPase A,

it has been suggested that an intermolecular hydrogen bondbetween tyrosine-248 and a neighboring CPase A molecule inthe crystal is responsible for the low activity ofCPase A crystals(22). This interpretation needs to be tested for the crystals ofCPase Al used in these experiments, but it is clearly not truefor the crystals used in the x-ray studies. As show in Fig. 6, theclosest intermolecular contact of the tyrosine-248 phenol oxy-

5j~.8

r FIG. 5. Binding of residues 36-39 of PCI (thicklines) to the active site of CPA. Hydrogen bonds be-tween tyrosine-248 and valine-38 are indicated bydotted lines.

.1

I

Proc. Nad Acad. Sci. USA 78 (1981)

Proc. NatL Acad. Sci. USA 78 (1981) 5459

FIG. 6. Intermolecular contacts of tyrosine-248in the x-raycrystals. Threonine-14 and tyrosine-248 were omitted from thestructure factor calculation from which this difference map at1.5 A resolution was drawn in order to obtain an unbiased elec-tron density map. Coordinates for threonine-14 and tyrosine-248 are superimposed on the electron density.

gen is with the methyl group of threonine-14, 3.8 A away. Thephenol oxygen of tyrosine-248 is hydrogen bonded to a watermolecule.

Studies on AAT-CPase A crystals also support the conclusionthat tyrosine-248 has free mobility in the x-ray crystals. At pH8.2, AAT-CPase A is red both in solution and in the x-ray crys-tals, indicative of an AAT-Zn interaction, whereas crystals ofAAT-CPase A (Anson) are yellow, indicative of an unchelatedAAT group (4). Based on comparison of the solution and crystalbehavior of AAT-CPase A (Anson), Vallee and coworkers (6,18-22) suggested that the solution and crystals conformationsof CPase A are different. As shown by Quiocho et aL (4) how-ever, the appropriate examination of the solution and crystalbehavior of AAT-CPase A (Cox) demonstrated that the AATgroups are in similar environments in both states. These resultsonce again stress the necessity of distinguishing between thevarious crystal forms of CPase A when interpreting experimen-tal results.

Summary. We make the following observations.(i) Because the crystals that we study show about one-third

of the activity they have in solution, we have a functional en-zyme in our crystals. The most readily available commercialcrystalline form is less active by a factor of about 100.

(ii) Different sites for hydrolysis of peptides and esters arenot required, provided entry into the active site pocket is ratelimiting for peptides but precedes the rate-limiting catalyticsteps for esters. Thus, IAA, which binds at S'j, can inhibit com-petitively for esters and noncompetitively for peptides. Ofcourse, different detailed mechanisms may apply to membersof these two classes even though cleavage occurs between S,and S', for both classes.

(iii) At pH 7.5, the percentage oftyrosine-248 anion-Zn com-plex in the absence of substrate is most probably zero and iscertainly <10% in our 1.5 A refinement. It is therefore likelythat the arsanilazo tyrosine-248-Zn interaction is a property ofthis arsanilazo derivative ofCPase A and that a tyrosine-248-Zninteraction occurs in low percentage, if it occurs at all, in thenative enzyme. Activation of tyrosine-248 by Zn is thereforeunlikely.

(iv) Inhibitors such as IAA might also bind noncompetitivelyto the enzyme in solution near tyrosine-198, phenylalanine-279,and arginine-71. Intermolecular contacts in our crystal formmay reduce binding near this "recognition" site.

(v) The binding occupancies of the ligands described in thispaper are between 28% and 55%. Ligand binding to CPase Ain our active crystals is accompanied by large negative electron

density -at the original position of tyrosine-248 in the native en-zyme. Thus, the binding of ligands to the enzyme induces theconformational change in tyrosine-248 described in the earlierx-ray diffraction studies.

This work has been supported by the National Institutes of HealthGrant GM 06920; National Science Foundation Grant PCM-77-11398supported the computational facilities.

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