Proc. Natl. Acad. Sci. USAVol. 83, pp. 7568-7572, October 1986Biochemistry

X-ray crystallographic investigation of substrate binding tocarboxypeptidase A at subzero temperature

(protein crystallography/x-ray cryoenzymology/enzyme-substrate complex)

DAVID W. CHRISTIANSON AND WILLIAM N. LIPSCOMBGibbs Chemical Laboratories, Department of Chemistry, Harvard University, Cambridge, MA 02138

Contributed by William N. Lipscomb, May 22, 1986

ABSTRACT A high-resolution x-ray crystallographic in-vestigation of the complex between carboxypeptidase A (CPA;peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) and the slowlyhydrolyzed substrate glycyl-L-tyrosine was done at -9°C.Although this enzyme-substrate complex has been the subjectof earlier crystallographic investigation, a higher resolutionelectron-density map of the complex with greater occupancy ofthe substrate was desired. All crystal chemistry (i.e., crystalsoaking and x-ray data collection) was performed on a diffrac-tometer-mounted flow cell, in which the crystal was immobi-lized. The x-ray data to 1.6-A resolution have yielded awell-resolved structure in which the zinc ion of the active siteis five-coordinate: three enzyme residues (glutamate-72, histi-dine-69, and histidine-196) and the carbonyl oxygen and aminoterminus of glycyl-L-tyrosine complete the coordination poly-hedron of the metal. These results confirm that this substratemay be bound in a nonproductive manner, because thehydrolytically important zinc-bound water has been displacedand excluded from the active site. It is likely that all dipeptidesubstrates of carboxypeptidase A that carry an unprotectedamino terminus are poor substrates because of such favorablebidentate coordination to the metal ion of the active site.

The zinc metalloenzyme carboxypeptidase Aa (CPA;peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) is anexopeptidase that exhibits preferred specificity toward pep-tides or esters bearing large hydrophobic carboxyl-terminalresidues such as phenylalanine. The proteolytic andesterolytic mechanism(s) of this protease have been thesubject of a wealth of chemical and structural study that hasbeen recently reviewed (1-4). Two possible hydrolytic mech-anisms include the attack of water/incipient hydroxide di-rectly at the scissile carbonyl carbon of the substrate,promoted by glutamate-270 and/or zinc, or the attack of they-carboxylate of glutamate-270 at the scissile carbonyl car-bon, with subsequent formation of a mixed anhydride inter-mediate, which then undergoes hydrolysis to yield products.Either the zinc ion of the active site or the positively chargedguanidinium moiety of arginine-127, or both, may serve topolarize the substrate carbonyl prior to its attack (regardlessof the general mechanism). Mechanistic analyses have been-aided in part by structural studies of the native enzyme (5-7)and its complexes with different inhibitors (8-15), includingthe slowly hydrolyzed (16) substrate glycyl-L-tyrosine (GY;Fig. 1) (6, 7), which is a competitive inhibitor ofmore rapidlycleaved substrates. Such nonacylated dipeptides are typical-ly cleaved more slowly by a factor of 1000-5000 than theiracylated analogues (17). The principal features of the mostrecent (6) structural study of the CPA-GY complex at 2.0-Aresolution (using x-ray data collected at 4°C) were that itscarbonyl oxygen coordinated to zinc at a position different

OH

CH2

H3N-CH2-_-NH-C -CO0

FIG. 1. The slowly hydrolyzed CPA substrate glycyl-L-tyrosine.This compound is a competitive inhibitor of more rapidly cleavedsubstrates; although it is depicted as the neutral zwitterion, bothchemical studies and the results of the current crystallographicinvestigation suggest that it binds to carboxypeptidase A as theanion-i.e., with the terminal amino group in the free base form.

from that of the native zinc-bound water/hydroxide and thatthe terminal amino group statistically occupied two positions:(i) coordinated to zinc (as the free base), and (ii) hydrogen-bonded to glutamate-270 (possibly as a quaternary ammoni-um cation). The terminal carboxylate formed a salt link witharginine-145, and tyrosine-248 was in the "down" confor-mation, presumably donating a hydrogen bond to one of theterminal carboxylate oxygens.The current high-resolution x-ray crystallographic inves-

tigation of the CPA-GY complex was performed at subzerotemperature in order to further retard the slow enzyme-catalyzed hydrolysis reaction in the crystal. An aqueous-organic cryosolvent buffer system was used in order to allowfor subzero temperatures at the crystal. Because the crystalform ofCPA used in this study exhibits one-third the activityofthe enzyme in solution (18), a diffractometer-mounted flowcell assembly was used to provide a continuous, fresh supplyof GY to the enzyme crystal, in case appreciable amounts ofGY could be cleaved and released by the enzyme over thetime span of crystallographic data collection. X-ray datacollected at -90C to 1.6-A resolution conclusively reveal thestructure of an intact enzyme-substrate complex.

MATERIALS AND METHODSCPA and GY were purchased from Sigma and used withoutfurther purification. CPA was crystallized in space groupP21 (a = 51.60 A, b = 60.27 A, c = 47.25 A, /3 = 97.27 A,crystal habit elongated along the a axis), by dialysis of theenzyme (solubilized in 1.2 M LiCl/0.02 M Tris-HCl, pH 7.4)against 0.2M LiCl/0.02M Tris-HCl, pH 7.4, at 40C. The CPAcrystals were slightly crosslinked (19) with 0.2% (vol/vol)glutaraldehyde for 6 hr following transfer to 0.1 M LiCl/0.02M barbital-LiOH, pH 7.5. Crystals were mounted in quartzcapillaries that, in turn, were fastened to a diffractometer-

Abbreviations: CPA, carboxypeptidase Aa; GY, glycyl-L-tyrosine;Me2SO, dimethyl sulfoxide.

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Proc. Natl. Acad. Sci. USA 83 (1986) 7569

mounted yoke adapted from the design of Wyckoff andcolleagues (20). The crystal was secured within the capillaryby pipe-cleaner fibrils and Sephadex. The use of Sephadex inthe vicinity of the crystal facilitated initial optical alignmentin the x-ray beam. Polyethylene tubing of the appropriatediameter was attached to each end of the yoke-boundcapillary, allowing for a continuous flow of buffer solutionpast the immobilized enzyme crystal. The flow cell assemblyis illustrated in Fig. 2.A continuous stream of buffer solution (-5 ml/day) passed

through the flow cell and was gradually changed, withdecreasing temperature, to a cryosolvent mixture consistingof 40% (vol/vol) dimethyl sulfoxide (Me2SO)/0.15 MLiCl/0.02 M cacodylic acid-LiOH, pH 7.4. This particularmixture was chosen for several reasons: (i) of several organicco-solvents tested, Me2SO does not bring about physical orcrystallographic disorder to CPA crystals, even at roomtemperature; (ii) aqueous Me2SO solutions display a dielec-tric constant very near that of pure water; and (iii) theprotonic activity of cacodylic acid buffer solutions is nearlyinsensitive to temperature variation. Fink and Petsko (21)have recently reviewed general methods in x-ray cryoenzy-mology, and Douzou and colleagues (22) have compiled thephysical and chemical constants of various mixed cryosolv-ent systems and the protonic activities of various bufferreagents as a function of temperature. An FTS Systems(Stone Ridge, NY) XR-85 air jet crystal cooler was adaptedto the diffractometer-mounted Syntex (Nicolet; Madison,WI) LT-1 cold air stream delivery system. The temperatureinside the flow cell was checked periodically with a Sensortek(Clifton, NJ) IT-23 0.009-inch copper/constantan thermo-couple, which easily fits inside the 0.7-mm quartz capillary.

After the system was equilibrated at -250C, intensity datawere collected from one crystal to a resolution of 2.0 A andprocessed as described (12). Data collection was performedon a Syntex (Nicolet) P21 four-circle automated diffractom-eter using procedures described elsewhere (12). Initial dif-ference electron-density maps calculated against the native

polyethylenetubing

tape

FIG. 2. Design of the diffractometer-mounted flow cell. The flowinto the quartz capillary arcs clockwise over the top of the assembly,since this "precools" the buffer solution before it actually reachesthe crystal. Flow rates employed were usually very slow (ca. 5

ml/day) to prevent movement of the crystal.

enzyme revealed the presence of a zinc-bound Me2SOmolecule that had displaced the zinc-bound water of thenative enzyme. No other molecules ofMe2SO were observedto bind to the enzyme or displace enzyme-bound solventwater molecules. Building of Me2SO into the maps was doneon an Evans and Sutherland PS300 interfaced with a VAX11/780, with graphics software (FRODO) developed by Jones(23). Reciprocal space least-squares refinement (24) con-verged smoothly to a final crystallographic R factor of 0.162at 2.0-A resolution, where R is YIIF01 - JFJ l/E F0j, and IF01and IFJl are observed and calculated structure factors, re-spectively.Another crystal was mounted in the flow cell and equili-

brated at -90C in a cryosolvent mixture consisting of 20%(vol/vol) Me2SO/0.15 M LiCl/0.02 M cacodylic acid-LiOH,pH 7.4. This solution was then gradually changed to one ofsimilar composition plus 0.1 M GY. This mixture was allowedto flow past the immobilized crystal for 1 day before datacollection. It must be stressed that at no time was the crystalexposed to the substrate at higher temperatures; all "crystalsoaking" was performed at -9°C within the diffractometer-mounted flow cell prior to data collection. Thus, the hydrol-ysis reaction, albeit slow for this particular substrate, wassignificantly hindered. Additionally, the continuous flow offresh GY past the crystal throughout data collection helpedto ultimately ensure the observation of an intact complex: incase a molecule ofGY was cleaved, another one could comealong and take its place, assuming that substrate and productswould be free to diffuse through the solvent channels in thecrystal.

Intensity data were collected from two crystals under theseconditions to a resolution of 1.6 A. These data were pro-cessed as described (12) and used in the calculation ofdifference electron-density maps against the native enzyme.Initial maps revealed an intact CPA-GY complex; the GYmolecule was therefore built into the difference density, andthe enzyme-substrate model was refined against the ob-served data. Crystallographic refinement converged to a finalR factor of 0.178 at 1.6-A resolution. A difference electrondensity map calculated with Fourier coefficients IFOI - IFCIand phases calculated from the final model coordinatesshowed rms residual electron density (a) of 0.07 e/A3; thehighest peaks in the vicinity of the active site were just under3a. No peaks corresponding to any residual zinc-boundMe2SO were observed in this map. An rms error in atomicpositions was estimated to be about 0.2 A based on relation-ships derived by Luzzati (25), which attribute all discrepan-cies between |FOI and |FCI to errors in atomic coordinates. Assuch, the error of ca. 0.2 A probably represents an upperlimit, since discrepancies between observed and calculatedstructure factors can also be attributed to a host of otherexperimental conditions, as well as methods of data collec-tion and reduction.

RESULTS AND DISCUSSIONThe structure of CPA in the unique environment afforded bythe aqueous/organic cryosolvent and the subzero tempera-ture is remarkably similar to that ofthe native enzyme. Basedupon data collected at -25°C in a cryosolvent mixtureconsisting of 40% Me2SO, only one significant difference isobserved: a molecule ofMe2SO coordinates to the zinc ion ofthe active site under these conditions, and it displaces thezinc-bound water of the native enzyme in doing so. NoMe2SO molecules are observed to displace the water mole-cules normally residing in the hydrophobic pocket of thenative enzyme. Furthermore, no appreciable temperature- orsolvent-dependent conformational changes of enzyme resi-dues are observed. A difference electron-density map of the

Biochemistry: Christianson and Lipscomb

7570 Biochemistry: Christianson and Lipscomb

CPA-Me2SO complex is presented in Fig. 3. The prominentfeature of this map is a very dense peak corresponding to thezinc-bound Me2SO molecule, with a zinc-oxygen distance of2.2 A. This distance is in agreement with that of theMe2SO-zinc interaction ofhorse liver alcohol dehydrogenasein its Me2SO complex with the NADH holoenzyme (26). Itshould be noted that the distance of the Me2SO sulfur fromzinc, 3.4 A, and also the orientation of the pyramidal-likeMe2SO so that the lone electron pair of sulfur is directed awayfrom the active site, preclude a zinc-sulfur bond. Also, nochanges are observed in the conformations of enzyme resi-dues serving as zinc ligands, nor does the zinc itself moverelative to these residues [the movement of zinc upon thebinding of larger inhibitors has been discussed (15)]. Themechanism of CPA inhibition by Me2SO likely involvesMe2SO coordination to zinc with resultant exclusion of thecatalytically important water molecule from access to thereactive carbonyl of the substrate.When the cryosolvent system is altered to 20% (vol/vol)

Me2SO and 0.1 M GY at -9°C, GY is observed bound to theactive site intact. Furthermore, no trace of residual zinc-bound solvent remains. A difference electron-density map ofthis enzyme-substrate complex calculated at 1.6-A resolu-tion is presented in Fig. 4. The observed planarity of thedifference electron density of the glycine and the amidelinkage support this conclusion-if any zinc-bound Me2SO orwater remained at appreciable occupancy, the differencedensity of this portion of the substrate would be punctuatedby "bumps" rather than appearing consistent and flat. Thecoordination position ofMe2SO is different from that of GY;indeed, the two carbon atoms and the sulfur atom of Me2SOwould lie outside the electron density outlining the substratein Fig. 4. Additionally, there is no evidence for residualMe2SO in the final difference electron-density map calculatedwith Fourier coefficients 1F01 - IFcI and phases calculatedfrom the final model. No peaks in this map are evident nearthe coordination position of Me2SO even at the low level of1.5c. Hence, we conclude that it is solely GY that is observedbound to the active site of CPA, and the substrate is boundin an intact, unhydrolyzed condition. The glycine portion ofGY spans the zinc ion and chelates in bidentate fashion

through both the amide carbonyl oxygen and the terminalamino nitrogen (the O-Zn distance is 2.3 A, and the N-Zndistance is 1.9 A). It appears that it is solely the aminonitrogen that is responsible for displacing the formerlyzinc-bound solvent molecule; i.e., the coordination positionof the carbonyl oxygen would not require the displacement ofthe zinc-bound water of the native enzyme. The currentresults differ somewhat from those observed in the lower-resolution study (6), where the amino terminus appeared tobe disordered between zinc coordination and hydrogenbonding to glutamate-270; its position is now better definedgiven the higher resolution and greater occupancy (60%versus 30%) of the bound GY molecule. Additionally, thezinc ion is observed to move about 0.3 A in the directiontoward arginine-127. Such movement of the metal ion uponits interaction with enzyme-bound inhibitors has been dis-cussed (15). A stereoview of the CPA-GY complex ispresented in Fig. 5.

It is not surprising to find that the amino terminus of thebound dipeptide is not observed to exist as a quaternaryammonium cation; given all the other strong binding inter-actions between CPA and GY, the amino terminus finds itselfin the very electropositive environment of the zinc ion andnearby arginine-127. Although arginine-127 is not observed tohydrogen-bond to the bound substrate, its close proximity tothe bound substrate would certainly favor the free base formof the amino terminus by electrostatic considerations. Addi-tionally, Yanari and Mitz (27) have concluded, on the basisof the pH dependence of GY inhibition kinetics, that GYbinds to the enzyme as the anionic species-i.e., with theamino terminus as the free base. On the basis of electronparamagnetic resonance studies of the Co2+-substituted en-zyme and its complex with GY prepared in the presenceof 170-enriched water, Kuo and Makinen (28) have conclud-ed that the metal-bound water/hydroxide is displaced uponthe binding ofGY. This proposition is entirely consistent withthe observed mode of binding in the CPA-GY complex. It islikely that all dipeptide substrates of CPA bearing an unpro-tected amino terminus are poor substrates due to suchfavorable bidentate chelation to the zinc ion ofthe active site.This chelation may significantly compete with the catalyticevent(s). Since the hydrolytically important zinc-boundwater/hydroxide has been excluded from the active site in

K

+ +

+

726

FIG. 3. Portion of a difference electron-density map calculatedwith Fourier coefficients IF.1 - IF~j and phases calculated from therefined CPA-Me2SO model coordinates less the nonhydrogen atomsof the solvent molecule. Pertinent enzyme residues (glutamate-270,glutamate-72, histidine-69, histidine-196) are indicated by their se-

quence number; the zinc ion appears as a small black sphere. Activesite water molecules appear as crosses. The oxygen of the Me2SOmolecule is 2.2 A from the zinc ion; both the distance of the sulfurfrom zinc (3.4 A) and its orientation so that its lone electron pair facesthe opposite direction from zinc preclude a zinc-sulfur bond.

FIG. 4. Portion of a difference electron-density map calculatedwith Fourier coefficients 1F01 - IFJI and phases calculated from therefined enzyme-substrate model coordinates less the nonhydrogenatoms of the substrate. Pertinent enzyme residues (glutamate-270,glutamate-72, histidine-69, histidine-196, tyrosine-248) are indicatedby their sequence numbers; the zinc ion appears as a small blacksphere. Note the continuous and bifurcated electron density span-ning the zinc ion. The smoothness and consistency of this densitysuggest that the intact substrate is bound. Indeed, the densitycorresponding to the peptide unit ofGY is quite planar, indicative ofthe planar peptide linkage ofthe intact dipeptide. The amino terminusof GY is 1.9 A, and its carbonyl oxygen is 2.3 A from the zinc ion.

Proc. Natl. Acad. Sci. USA 83 (1986)

Proc. Natl. Acad. Sci. USA 83 (1986) 7571

Y248Y248

p7>

FIG. 5. Stereoview of GY bound to the active site of CPA. Enzyme residues are indicated by their standard one-letter abbreviations andsequence numbers-e.g., E, glutamate; H, histidine; R, arginine; Y, tyrosine.

the CPA-GY complex, it is likely that the binding modeobserved for GY in this enzyme-substrate complex repre-sents a nonproductive-i.e., catalytically inactive-state.Other binding interactions observed are typical of those

found in CPA-inhibitor complexes (8-15): the hydroxyben-zyl group of tyrosine resides in the hydrophobic pocket, orspecificity pocket, of the Sj' subsite, and the terminalcarboxylate is salt linked with the guanidinium moiety ofarginine-145. The phenolic residue of tyrosine-248 is in the"down" conformation, and its phenolic oxygen is 2.8 A awayfrom one of the terminal carboxylate oxygens of GY. Thisinteraction clearly favors the un-ionized state oftyrosine-248.The substrate lies in a slightly different position from thatdetermined in the previous low-resolution study (6); an rmsdifference in GY atomic positions was calculated to be 0.6 Abetween the prior unrefined coordinates and those obtainedfrom the current model refined at high resolution. Arginine-127, although having moved toward the bound substraterelative to its position in the native enzyme, is not withinhydrogen-bonding distance to the substrate carbonyl oxy en(the distance to the nearest guanidinium nitrogen is 4.3 A).This residue is observed to hydrogen-bond to the GYcarbonyl in its complex with the apoenzyme (29), as well ashydrogen-bond to the carbonyl of a substrate analogue in arecent x-ray crystallographic study (13). Finally, the y-carboxylate of glutamate-270 is within hydrogen-bondingdistance to the amide nitrogen of the peptide linkage, afeature that supports proposals involving glutamate-270 as aproton donor (12, 30). Selected distances of enzyme-sub-strate interactions are recorded in Table 1.A role for the positively charged guanidinium moieties of

the three arginine residues lining the active site cleft of CPAhas been proposed (31, 32) in view of the precatalyticassociation mechanism of substrate with enzyme. More

Table 1. Selected enzyme-substrate distancesProtein atom Substrate atom Distance, A

Arg-145 N1 Carboxylate 01 3.3tArg-145 N2 Carboxylate 02 3.OtTyr-248 phenolic 0 Carboxylate 01 2.8tGlu-270 OF' Amino N 3.6Glu-270 OE2 Amino N 4.6Glu-270 OE0 Carbonyl C 3.6Glu-270 0e2 Amide N 2.6tSer-197 carbonyl 0 Amino N 2.8tZinc Amino N 1.9Zinc Carbonyl 0 2.3Arg-127 N2 Carbonyl 0 4.3

tPossible hydrogen bond.

recently, the direct involvement of arginine-127 with boundinhibitors prompted speculation as to its possible involve-ment in actual catalytic steps mediated by the enzyme (1, 12,14, 15). Such a catalytic role was initially considered toinvolve the shift of the carbonyl of the putative anhydrideintermediate from zinc coordination to hydrogen bondingwith arginine-127 in order to facilitate deacylation by zinc-bound water/hydroxide (1). However, later structural stud-ies that modeled the intermediate of the promoted attack ofwater directly at the substrate carbonyl prompted speculationas to the role of arginine-127 in the initial binding of thescissile substrate carbonyl before any hydrolytic steps (12)and then to the stabilization of the oxyanion tetrahedralintermediate (14, 15). Such an effect, of course, could beachieved through both a hydrogen bond and/or electrostaticstabilization. Also, the initial step involving the polarizationof the substrate carbonyl, previously considered to be theexclusive role of the metal ion at the active site, could beshared with the positively charged side chain of arginine-127.Indeed, both the zinc ion and arginine-127 are oriented insuch a way as to provide optimal polarization of the scissilecarbonyl. An unidentified arginine residue is observed to beofcatalytic significance in the proteolytic mechanism, but notthe esterolytic mechanism, based upon chemical modifica-tion studies (33). A sequence ofbinding modes, with differentrate-determining steps for peptides and esters involvingarginine-127, may account for the observed kinetic anoma-lies, although it is not clear whether this chemical modifica-tion occurs at arginine-71, -127, or -145.The probably nonproductive mode of binding observed in

the CPA-GY complex does not involve a hydrogen-bondinginteraction with arginine-127. It is quite possible that theobserved bidentate GY may be bound solely because of thefavorable chelate interaction provided by the free aminoterminus; the involvement of arginine-127 in polarizing thesubstrate carbonyl is not precluded in a more catalyticallyfavorable productive mode of binding. This mode wouldinvolve simply the rotation of the peptide unit ofGY by about30-40° toward arginine-127. Similar roles for potentiallyelectropositive histidine residues in the active sites of therelated zinc proteases thermolysin (histidine-231) andpeptidase G (histidine-190) are also feasible on the basis ofthestructure of a thermolysin-inhibitor complex (34) or on thebasis of preliminary x-ray crystallographic data on the active-site geometry and proximal residues to the active-site zinc ionofpeptidase G (35). Nevertheless, ifa metal-bound hydroxideis the catalytically responsible nucleophile, then the coordi-nation of a substrate carbonyl would lessen the nucleophiliccapability of such a metal-bound species. That is, in the caseof CPA, the zinc- and/or glutamate-270-promoted hydroxide

Biochemistry: Christianson and Lipscomb

E2

7572 Biochemistry: Christianson and Lipscomb

would be a more potent nucleophile if the substrate carbonylinitially coordinated to arginine-127, with subsequent shift ofthe oxyanion tetrahedral intermediate from arginine-127 tozinc. Similar electrophilic roles emerge for histidine-231 ofthermolysin and histidine-190 of peptidase G.The assignment ofthe catalytically responsible nucleophile

is a task more difficult than that of assigning the identities toactive site electrophiles. Glutamate-270 has been proposed(7) to attack the scissile carbonyl of the substrate, withsubsequent formation of a covalent mixed anhydride inter-mediate, which then undergoes hydrolysis to yield products.This mechanistic proposal is supported, in part, by theobservation ofa five-coordinate metal ion in low-temperatureelectron paramagnetic resonance spectroscopic experimentswith Co-CPA (28). The five metal ligands were presumed tobe glutamate-72, histidine-69, histidine-196, a zinc-boundhydroxide, and a carbonyl of an anhydride intermediate.However, such five-coordination is also structurally consist-ent with intermediates encountered in other mechanisticpathways. If a promoted-water mechanism were followed, afive-coordinate metal complex would result if the tetrahedralintermediate were to become chelated in bidentate fashion tothe metal ion. A structural model of such a possibility isillustrated in the complex of CPA with a hydrated aldehydeinhibitor (12), where both oxygens ofthe tetrahedral gem-diolmoiety straddle the zinc ion. Furthermore, a zinc-promotedwater would be a much more potent nucleophile than woulda carboxylate-promoted water. Glutamate-270, i.e., thecarboxylate, may play an important role in this case as themediator of proton transfer to the leaving group of thecollapsing tetrahedral intermediate. As such, glutamate-270may act in concert with the zinc ion in the promotion of thehydrolytically important zinc-bound water/hydroxide. Theresultant tetrahedral intermediate would be exceptionallystabilized if it were coordinated in bidentate fashion to themetal ion, and even more so if the positively chargedguanidinium moiety of arginine-127 were to participate in thestabilization mechanism. Low-temperature studies will helpto elucidate the structure of the putative five-coordinateintermediate of CPA-catalyzed esterolytic, and quite possi-bly proteolytic, reactions.

We thank Mr. Peter David for his assistance in preparing thecrystals as well as his helpful discussions during the course of thiswork. The support ofW. R. Grace & Co. is gratefully acknowledged;additionally, D.W.C. thanks AT & T Bell Laboratories for a doctoralfellowship. Finally, we thank the National Institutes of Health forGrant GM 06920 in support of this research, and the National ScienceFoundation for Grant PCM-77-11398 in support of the computationalfacility.

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Proc. Natl. Acad Sci. USA 83 (1986)