Catalytic Conformation of Carboxypeptidase A

Tm JOURNAL OF BIOLDGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 6, Issue of February 11, pp. 4587-4595, 1994 Printed in U S A .

Catalytic Conformation of Carboxypeptidase A STRUCTURE OF A TRUE ENZYME REACTION INTERMEDIATE DETERMINED BY ELECTRON NUCLEAR DOUBLE RESONANCE*

(Received for publication, May 25, 1993, and in revised form, October 15, 1993)

Devkumar Mustafi and Marvin W. MakinenS From the Department of Biochemistry and Molecular Biology, The University of Chicago, Cummings Life Science Center, Chicago, Illinois 60637

The structure of a catalytically competent reaction intermediate of carboxypeptidase A (CPA) formed with the specific spin-label ester substrate 0-[3-(2,2,- S,S-tetramethyl-l-oxypyrrolinyl~propen-2-oyll-~-~- phenyllactate through application of cryoenzymological methods has been determined by electron nuclear double resonance (ENDOR) and molecular modeling. It is shown that the reaction intermediate is best identified as a mixed-anhydride acylenzyme derivative in which the side chain of Glu-270 is acylated by the spin-label substrate, in agreement with previous cryoenzymological and spectroscopic studies from this laboratory. From the observed proton ENDOR shifts corresponding to principal hyperfine coupling components and assigned by se- lective deuteration, the dipolar hyperfine coupling components were calculated to estimate electron-proton distances. With these ENDOR-determined distances as constraints, the conformation of the substrate free in solution and in the active site of CPAhas been determined on the basis of torsion angle search calculations. With a catalytically active, acetylated form of CPA, we have also assigned the position of the side chain of Tyr-198 with respect to the nitroxyl group. The positional assignments of both substrate and active-site residues in a true reaction intermediate provide important constraints in de- fining the structural basis of action of CPA.

Intermediates of enzyme-catalyzed reactions are short-lived and have been, in general, inaccessible to structure analysis by x-ray diffraction methods. While more than 50 different enzyme structures are currently known through x-ray studies at high to moderate resolution, crystallographic investigations have been confined almost entirely to unliganded enzymes or stable enzyme-ligand (inhibitor) complexes. This circumstance reflects the essential structural difference between the interactions of substrates and enzymes in short-lived catalytically reactive states and of inhibitors and enzymes in stable nonproductive complexes. An inhibitor cannot elicit all of the interactions with an enzyme that are responsible for the catalytic conversion of substrate to product since the stability of enzyme- inhibitor complexes is due to their nonproductive spatial relationships. For this reason, it is necessary to find methods to determine the structures of true reaction intermediates.

* This work was supported by Grant GM 21900 from the National Institutes of Health. The costs of publication of this article were de- frayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed: Dept. of Biochemis- try and Molecular Biology, The University of Chicago, 920 E. 58th St., Chicago, Illinois 60637-1432.

Previous chemical, cryokinetic, and spectroscopic studies of carboxypeptidase A (CPA)I (peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) provide an indication of the potentially incisive results that can be obtained through a combined structural and cryoenzymological approach to characterize reaction intermediates of enzymes. For CPA, in particular, chemical and physi- cal studies of low temperature-stabilized reaction intermediates show that (i) proteolytic and esterolytic reactions proceed through a mixed-anhydride acylenzyme intermediate (1-41, (ii) the active-site metal ion becomes penta-liganded in the acylenzyme intermediate to accommodate both a water molecule (or hydroxide ion) and the carbonyl oxygen of the scissile bond (5, 6), and (iii) the metal-bound water is responsible for hydro- lytic breakdown of the acylenzyme (1, 6, 7). In contrast, the earlier results of x-ray studies of enzyme-inhibitor complexes (8-11) and of chemical and kinetic studies (12-16) had been invoked to suggest that (i) the reaction proceeds through a general base-catalyzed mechanism, (ii) the substrate displaces the metal-bound water, and (iii) the active-site metal ion remains tetra-liganded throughout the reaction. The essential difference in these contrasting interpretations derives simply from chemical identification and structural characterization of a true reaction intermediate of CPA.

In earlier cryoenzymological and spectroscopic studies of the acylenzyme reaction intermediate of CPA, structural analysis was limited to application of EPR spectroscopy to assign the active-site metal ion coordination environment (5, 6) and to model a spin-label substrate in the active site on the basis of the spectroscopically determined distance between the spin- label nitroxyl group and the metal ion in the Co2+-reconstituted enzyme (17). No direct information could be obtained at that time to assign the conformation of the bound substrate, the chemical origins of the tight binding of the substrate within the active site, or the structural relationships of active-site residues to the substrate. Such shortcomings can be overcome, as shown in the preceding study of a-chymotrypsin (18), by employing a combined approach of cryoenzymology (19-21) with a method of structure determination of high precision.

We have shown that electron nuclear double resonance (EN- DOR) spectroscopy, particularly of nitroxyl spin-labeled mol- ecules, can be incisively applied with an accuracy that is ex- ceeded only by that of single crystal x-ray diffraction methods (22-26). Here, we describe the ENDOR-determined structure of a catalytically competent reaction intermediate of CPA formed with the specific spin-label ester substrate 0-[3-(2,2,5,5-tetramethyl-l-oxypyrrolinyl)propen-2-oyl]-~-~-phenyllactate (TEP-

The abbreviations used are: CPA, carboxypeptidase A; Ac-CPA, acetylated carboxypeptidase A (in which tyrosinyl residues have been acetylated as described under “Experimental Procedures”); ENDOR, electron nuclear double resonance; TEPOPL, 0-[3-(2,2,5,5-tetramethyl- l-oxypyrrolinyl)propen-2-oyll-~-~-phenyllactate; ClCPL, 0-(trans-p- chlorocinnamoyl)-L-/3-phenyllactate.

4587

4588 Catalytic Conformation of Carboxypeptidase A

OPL). For structural characterization of the reaction intermediate of CPA, we have employed (i) cryoenzymological methods to accumulate the reaction intermediate in solution, (ii) EN- DOR spectroscopy to determine dipolar separations between the nitroxyl unpaired electron and surrounding nuclei for use as constraints in torsion angle search calculations, and (iii) molecular modeling to determine precise structural and stereo- chemical relationships of the substrate to active-site residues. To assign the positions of amino acid side chains in the active site, we have employed the acetylated form of CPA, in which the phenolic OH groups of the active-site residues Tyr-198 and Tyr-248 are modified (€427,281. The ENDOR-determined electron-proton distances define not only the conformation of the acyl moiety of the substrate in the active site, but also the relative positions of these active-site residues in a kinetically competent reaction intermediate of CPA.

EXPERIMENTAL PROCEDURES

Materials General-The parent spin-label 2,2,5,5-tetramethyl-l-oxypyrroline-

.3-carboxylic acid was obtained by hydrolysis of 2,2,5,5-tetramethyl-l- oxypyrroline 3-carboxamide (Aldrich) according to Rozantsev (29). 1,l'- Carbonyldiimidazole, p-chlorocinnamic acid, 3-phenylpropenoic acid, and L-p-phenyllactic acid were obtained from Aldrich. Deuterated solvents b99.5 atom % 2H) were obtained from Cambridge Isotope Labo- ratories, Inc. (Woburn, MA) or from Aldrich. Tetrahydrofuran was re- fluxed and distilled over lithium aluminum hydride and stored over molecular sieves. All other reagents have been described (18).

0-(trans-p-Chlorocinnamoylj-L-p-phenyllactate (CLCPL)-This com- pound was synthesized as described by Suh and Kaiser (30) (46% yield; m.p. 124-124.5 "C).

TEPOPL-This ester was synthesized by reaction of the acid chloride of 3-(2,2,5,5-tetramethyl-l-oxypyrrolinyl)-2-propenoic acid (31) with L-p-phenyllactic acid as described previously (32). The oily spin-label ester product was dissolved in a small volume of ethyl acetate, and an equimolar amount of potassium 2-hexanoate in butanol was added slowly with vigorous stimng. The resulting solution was then reduced in uacuo to a small volume, and ether was added until precipitation of the potassium salt of TEPOPL was complete. The overall yield was 27% (m.p. 165-167 "C) (analysis calculated (found): C, 60.58 (58.23); H, 5.85 (6.04); N, 3.53 (3.28); K, 9.86 (9.83)).

[3-2HlTEPOPL-This deuterated analog was synthesized as described above for TEPOPL, but with the use of 3-(2,2,5,5-tetramethyl- l-oxypyrrolinyl)-2-[3-2Hlpropenoic acid (31) as the starting material. Comparable results for melting point determination and elemental analysis were obtained as for TEPOPL.

N-Acetylimidazole-To 60 ml of freshly distilled tetrahydrofuran was added 4 ml (0.070 mol) of acetic acid. The solution was cooled to 0 "C, and 10 g of 1,l'-carbonyldiimidazole (0.062 mol) were added over 1 h with vigorous stirring. The mixture was further stirred for 1 h at 0 "C and brought to room temperature over 2 h. The solution was evaporated to dryness in uacuo. To the residue was added -100 ml of tetrahydrofuran, whereupon removal of the tetrahydrofuran by evaporation in uacuo produced a yellow-white crude product (m.p. 93-98 "C). m e r recrystallization five times from isopropenyl acetate, 5.4 g of pure N- acetylimidazole (79% yield) were obtained with a constant melting point of 102.7-103.5 "C (analysis calculated (found): C, 54.54 (53.32); H, 5.49 (5.30); N, 25.44 (25.23)). The mass spectrum of the product showed that the highest molecular weight species corresponded to an mle ratio of 110. N-PHJAcetylimidazole-For this analog, the same procedure was

followed, except that [2H,]acetic acid b99.5 atom % 2H; Cambridge Isotope Laboratories, Inc.) was used. The yield was 5.2 g (74% yield) of pure product (m.p. 102.5-103.5 "C) (analysis calculated (found): C, 53.08 (52.95); H, 5.35 (5.28); N, 24.76 (24.80)). The mass spectrum showed an mle ratio of 113, expected for the highest molecular weight species, with no evidence of a molecular ion peak at mle 110.

CPA and Acetylated Carboqypeptidase A (Ac-CPAtCrystalline bo- vine pancreatic CPA, prepared according to the method of Cox et al. (33), was obtained from Sigma and used as previously described (1, 32). Protein concentrations were determined on the basis of a molar absorptivity coefficient of 6.42 x lo4 M - ~ cm" at 278 nm for native CPA. Acetylation of the enzyme with acetylimidazole or [2H,lacetylimidazole was performed according to the method of Simpson et al. (27) by adding

0' II

C 3 = C 2 - C 1 - O - C a - C O O H

SCHEME 1.

a 10-12-fold excess of the reagent over -30 min to a solution of CPA in 2 M NaCl buffered with 0.03 M sodium cacodylate to pH 7.5 a t 23 "C. The reaction was quenched by cooling, followed by exhaustive dialysis against the same buffer at 4 "C. In control experiments, 3-phenylpropionic acid was used as a protective agent (28). In these experiments, CPA was first treated with an - 15-fold excess of 3-phenylpropionic acid, followed by acetylation and removal of reagents by dialysis. The inhibitor 3-phenylpropionic acid prevents acetylation of active-site tyrosyl residues (27, 28).

Methods Enzyme Kinetics-The steady-state kinetic parameters kcat and

k,,JKM were determined on the basis of initial velocity data collected spectrophotometrically and calculated on the basis of the change in absorptivity at 310 nm for ClCPL and at 274 nm for TEPOPL as described earlier (1, 17).

EPR and ENDOR-For preparation of ENDOR samples, perdeuter- ated organic cryosolvents and ZHzO buffer were used throughout. CPA and Ac-CPA were dialyzed against several changes of 2Hz0 buffer at 4 "C for -72 h. The procedure for preparing EPR samples at -60 "C using cryosolvents has been described previously (1, 2, 5, 6). Alterna- tively, samples were prepared in EPR tubes at -20 "C (CClJsolid COz bath) with the enzyme buffered to pD 7.52 with 0.03 M cacodylate in 4 M NaCI. First, the enzyme solution in the EPR sample tube was brought to -20 "C, and then a small aliquot of substrate in [2H,]methanol was injected through a plastic catheter into the sample tube. Mixing required no more than 15-30 s. In the EPR tube, the total sample volume was 240 pl, and the final solvent composition was -90% 2Hz0 buffer, 10% [2H,lmethanol (v/v). Typical concentrations of enzyme and substrate were 1.5 x lo-, and 1.0 x lo-, M, respectively. All samples were stored frozen in liquid nitrogen. EPR and ENDOR spectra were recorded as described in the preceding paper (18).

Molecular Modeling-The atomic numbering scheme of TEPOPL is shown in Scheme 1 for purposes of discussing molecular modeling results. Atomic coordinates for non-hydrogen atoms of TEPOPL were obtained from x-ray-defined molecular fragments of 2,2,5,5-tetramethyl-1-oxypyrroline 3-carboxamide (34) and L-p-phenyllactic acid (351, with the propenoyl moiety being added according to standard bond lengths and valence angles (31). Positions of hydrogen atoms were calculated as previously described (26,31). The atomic coordinates of CPA refined at 1.54-A resolution and of the inhibitor glycyl-L-tyrosine in the active site were obtained from the Brookhaven Protein Data Bank (36, 37). For molecular modeling of Ac-CPA, the x-ray structure of 2-acetoxy- 3-methylbenzoic acid (38) was employed to construct Tyr-198 and Tyr- 248 into acetyl tyrosinate residues.

FRODO (39) and INSIGHT (40) running on an Evans & Sutherland Molecular modeling was carried out with the use of the programs

PS390 graphics terminal. Application of the program package SYBYL (41, 42) for torsion angle search calculations is described in the preceding paper (18) and elsewhere (22-26, 31).

relationships pH = pH* and pD = pH* + 0.4 were used. pH* corresponds For studies involving normal and deuterated water as solvents, the

to the operational pH obtained with a glass electrode standardized in an ordinary water buffer solution. We have also corrected the pH for mixtures containing 14% (v/v) MeOH at -20 "C (19). For preparing ENDOR samples in an H,O/MeOH (86:14, v/v) cosolvent mixture at -20 "C buffered with sodium cacodylate, the pH adjustment corresponds to +0.2 units. In this study, the pH or pD was always adjusted to the value of 7.5.

Catalytic Conformation of Carboxypeptidase A 4589

TABLE I Comparison of kinetic parameters governing the hydrolysis of

TEPOPL and CZCPL catalyzed by CPA and Ac-CPA

Enzyme Substrate kcat KM

S-1

CPA TEPOPL 1.9 f 0.1 7.8 f 0.6 ClCPL

Ac-CPA TEPOPL 68.6 f 4.6 9.6 f 0.6 1.1 2 0.1 7.3 * 0.8

ClCPL 34.0 2 4.0 9.2 0.7

XlW M

RESULTS AND DISCUSSION

Kinetic Studies of Hydrolysis of TEPOPL Catalyzed by CPA and Ac-CPA

Kinetic parameters governing the hydrolysis of TEPOPL and ClCPL catalyzed by CPA and determined on the basis of initial velocity data are compared in Table I. The values of kcat show that the spin-label substrate is considerably less reactive to- ward hydrolysis than the classical ester substrate ClCPL, whereas the values of KM are essentially identical. Nonethe- less, the value of the specificity constant k,,& (-io4 M - ~ 5-l)

for TEPOPL is comparable to that of other specific peptide and ester substrates of CPA (7, 8). Also listed in Table I are the kinetic parameters of the two substrates with Ac-CPA, which we have employed in ENDOR studies. In this form of the enzyme, 4 tyrosyl residues are modified, including Tyr-198 and Tyr-248 in the active site (8, 27, 28). The kinetic results show that acetylation of CPA results in an -2-fold decrease in the value of kcat for both TEPOPL and ClCPL, while the value of KM remains unchanged. Similar observations for hydrolysis of 0-(trans-cinnamoy1)-L-P-phenyllactate by CPA and Ac-CPA were reported by Glovsky et al. (43). In control experiments using enzyme that had undergone acetylation in the presence of the competitive inhibitor 3-phenylpropionic acid, kinetic parameters for hydrolysis of both substrates were identical to those of the unmodified enzyme.

Substrate hydrolysis by CPA is controlled through a multi- point cooperative process that involves three separate regions of the active site: (i) the hydrophobic pocket that binds the COOH-terminal side chain of the substrate together with Arg- 145 responsible for binding the COOH-terminal carboxylate group; (ii) the bond cleavage site containing the active-site metal ion, its coordinating ligands, and the nucleophilic carboxylate side chain of Glu-270; and (iii) amino acid residues known as sites of secondary substrate recognition (44, 45). These regions are schematically illustrated in Fig. 1. Kinetic studies have shown that in CPA, the interaction of the S1' subsite with the COOH-terminal residue determines the value of KM and is dependent on the structure of the COOH-terminal side chain of the substrate, whereas the steric interactions of the Sz subsite with the substrate determine the value of kcat (7, 45). These relationships are reflected in the results of steady- state kinetic studies for hydrolysis of TEPOPL and ClCPL. Both substrates have identical COOH-terminal residues and exhibit identical KM values; however, their kcat values differ, reflecting their respective steric interactions with subsites of secondary substrate recognition, particularly in the Sz subsite. For both substrates, the values of KM are similar for CPA and Ac-CPA, indicating that chemical modification of the active-site residues Tyr-198 and Tyr-248 does not alter binding specificity.

Assignment of ENDOR Resonances and Estimation of Electron-Proton Distances

ENDOR of Reaction Intermediate of CPA Formed with TE- P O P G T h e EPR spectra of nitroxyl spin-labels are well char- acterized in terms of g and hyperfine interactions (46-48). Mi- crowave power saturation of the low field and central

N

FIG. 1. Schematic illustration of binding interactions of an oli- gopeptide substrate in the active site of CPA The active site com- prises five subsites (S,-S, and SI') located on both sides of the site of bond cleavage. Each subsite accommodates 1 amino acid residue of the substrate, and the positions Pi of the substrate are counted from the site of bond cleavage designated by the arrow.

absorption features of the EPR spectrum of spin-labels in frozen solution (termed settings A and B, respectively) selects different molecular orientations for ENDOR. The stratagem of employing the magnetic field dependence of the EPR spectrum of nitroxyl spin-labels for selection of molecular orientation in ENDOR has been described by us previously (22-26, 31). The dipolar hyperfine coupling components obtained from ENDOR then yield electron-proton separations and relative nuclear coordinates (cf Ref. 23) since the unpaired electron of spin-labels behaves as an effective point dipole, located at about the mid- point of the N-0 bond of the nitroxyl group (49).

In Fig. 2, we compare the proton ENDOR spectrum of the reaction intermediate of CPA formed with TEPOPL to those of the free substrate and of the products of hydrolysis. In this case, we have employed 4 M NaCl as solvent for accumulation of the reaction intermediate under conditions of subzero temperatures. Vallee and co-workers (14, 15) have similarly employed near-saturated solutions of NaCl for detecting reaction intermediates formed with Co2+-reconstituted CPA. This cryosolvent is ideal for CPA in view of its low viscosity compared to organic-aqueous cosolvent mixtures at subzero temperatures (19) and in view of the circumstance that the catalytic activity of CPA is maximized under conditions of high salt (7,501. The half-life of the reaction intermediate formed with TEPOPL is estimated to be -4 min at -20 "C on the basis of the observed value of kcat (Table I) at 25 "C, the enthalpy of activation of -17 kcal/mol, and the solvent kinetic isotope effect of -2.0 in kc,, determined in earlier studies (1, 17). The half-life of the intermediate is thus large compared to the mixing time of <30 s required for sample preparation and quenching of the reaction by freezing. The spectrum of the intermediate in Fig. 2 is observed only under conditions of enzyme in excess.

In Fig. 2, the specifically deuterated analog [3-2H]TEPOPL was used to facilitate assignment of the resonances of H-2 and estimation of ENDOR shifts. In Fig. 2, we have indicated the change in line splittings for the conversion of free substrate to reaction intermediate by a dashed stick diagram. Although the change in line splitting was small, as expected for a conjugated olefinic group, this change was precisely reproducible with formation of the reaction intermediate in 4 M NaCl or in the organic-aqueous cosolvent mixture of 40% MeOH, 40% H20,


-0.8 -0.4 0 0.4 0.8 MHz

FIG. 2. Proton ENDOR spectra of reaction mixture of [3-%]TE- POPL with CPAwith magnetic field at setting B. The top spectrum is for the substrate ( S ) free in solution; the middle spectrum is for the reaction intermediate ( I ) stabilized at -20 "C; and the bottom spectrum is for reaction products ( P ) and was taken after 3 h of incubation of the reaction mixture at 0 "C. The enzyme and substrate concentrations were 1.53 x and 1.22 x M, respectively. See "Experimental Procedures" for a detailed description of the preparation of samples for ENDOR. With I&, at setting B, both parallel and perpendicular hyperfine coupling components are observed, as identified by the stick diagram for H-2. The line splittings for H-2 of the substrate and product are indicated by the solid stick diagram, and slightly smaller line split-

dashed stick diagram. tings for the same proton of the intermediate are indicated by the

20% ethylene glycol (v/v) at -60 "C employed in earlier EPR studies (5, 6 , 17). On the other hand, the ENDOR shift of H-2 in the product remained identical to that in the free substrate in both solvent systems. Corresponding changes in line splittings for H-3 were also observed for the reaction intermediate formed with natural abundance substrate.

The observation that the ENDOR shift of H-2 was identical for the reaction intermediate in both solvent systems and distinguishably different from that in the free substrate or product is particularly important. The organic-aqueous cryosolvent mixture at -60 "C is significantly more viscous than the purely aqueous medium of 4 M NaCl at -20 "C (19) and therefore could retard mixing significantly. These results thus confirm that for the reaction intermediate in Fig. 2, the paramagnetic spin-label is entirely enzyme-bound. Since breakdown of the intermediate is rate-limiting under these conditions (1,171, we conclude that all of the bound spin-label substrate has been converted from the Michaelis complex into the reaction intermediate.

The values of the principal hyperfine coupling components for H-2 and H-3 and their corresponding electron-proton distances are summarized in Table I1 for the free substrate, reaction intermediate, and spin-labeled propenoate reaction product. In addition to the change in H-2 and H-3 ENDOR line splittings observed for formation of the reaction intermediate with the use of the 4 M NaCl solvent system, characteristic changes in the ENDOR spectrum were observed with corresponding precise reproducibility in the free matrix region cen- tered at the Larmor frequency (Av = 0). The spectra in this

TABLE I1 Summary of hyperfine coupling components and estimated

electron-proton distances in TEPOPL and its reaction intermediate formed with CPA and Ac-CPA

Proton AII AL Ai, AI? ALD ra

MHz A TEPOPL

H-2 1.413 0.798 -0.061 1.474 -0.737 4.75 = 0.02 H-3 1.211 0.811 -0.137 1.348 -0.674 4.91 = 0.02

Reaction

H-2 intermediate

H-3 1.383 0.790 -0.066 1.449 -0.724 4.78 2 0.02 1.225 0.821 -0.139 1.364 -0.682 4.88 = 0.02

dipolar hyperfine coupling components AllD and ALD can be calculated a From the observed hyperfine coupling components All and A,, the

under the constraintsAll> 0 >A, and (All + 2A l) = 3Ai, (22-26,31). The

kHz due to the line width of each absorption is included in the calcu- uncertainty in the observed hyperfine coupling components of 10-20

lation of electron-proton distances ( r ) using the classical dipolar equa- tion (23).

frequency region are illustrated in Fig. 3. It is seen that the shape of the spectrum of the reaction intermediate is distinguishably different from that of the free substrate. Further- more, if the tightly bound inhibitor L-benzylsuccinate (ICi < M) (51) is added first to the enzyme prior to addition of substrate, the spectrum corresponds essentially to that of the free substrate. On the other hand, if TEPOPL is added first to CPA followed by inhibitor, the spectrum remains that of the reaction intermediate.

These results confirm earlier observations made on the basis of difference ultraviolet absorption and EPR studies for the reaction intermediate in viscous cryosolvent mixtures (1, 5, 6, 17). These results also demonstrate that the inhibitor and substrate are bound in the same pocket of the enzyme and that the inhibitor cannot displace the acyl portion of the substrate from the enzyme once the reaction intermediate has been formed. It could be argued in the case of viscous organic-aqueous cosol- vents employed in earlier studies that the substrate is tightly bound in the active site as a noncovalent complex with the enzyme because of a long off-rate constant induced through the high viscosity of organic-aqueous cosolvent mixtures at low temperatures. This circumstance would hinder displacement by L-benzylsuccinate. Since a noncovalently bound substrate should be displaced by L-benzylsuccinate, particularly in the 4 M NaCl solution of low viscosity, the ENDOR results confirm our earlier interpretations that the reaction intermediate of TEPOPL with CPA is best rationalized as a mixed-anhydride acylenzyme species in which the acyl moiety is covalently attached to the carboxylate group of the side chain of Glu-270 (2, 17). This interpretation is confirmed by a detailed inspection of the ENDOR resonance features in the matrix region of the spectrum.

In Fig. 3, the well resolved ENDOR features indicated by stick diagrams for the free substrate are assigned to parallel and perpendicular hyperfine coupling components on the basis of ENDOR spectra collected at Ho settings A and B. For the free substrate, these resonance features must belong to Ha and H@i,z of the phenyllactic acid moiety since the protons of the phenyl group produce broad resonance features that are not resolved because of their distance and intrinsic line width, as has been noted for spin-labeled L-tryptophan (24) and L-phe-. nylalanine (25). Furthermore, these resonance features yielded splittings comparable in value and of similar spectral shape to the ethyl protons of ethyl 3-(2,2,5,5-tetramethyl-l-oxypyrrolinyl)-2-propenoate (26). On the other hand, for the reaction intermediate, the ENDOR splittings estimated as 0.248 and 0.125 MHz for All and Al, respectivtly, yielded an averaged electron-proton distance of 8.6 -c 0.6 A. Most important, how-


I rA*-

then Inhibitor

FIG. 3. Matrix proton ENDOR spectra of TEPOPL reacted with CPA in presence or absence of the biproduct inhibitor analog benzylsu succinate. The ENDOR spectra are shown for the following

termediate stabilized at -20 "C; mixture resulting from addition of the samples in top-to-bottom order: substrate free in solution; reaction in-

inhibitor to the enzyme solution first with thorough mixing at -20 "C, followed by addition of substrate; and mixture resulting from addition of substrate first to the enzyme solution with thorough mixing, followed by addition of the inhibitor. All samples were incubated for a total of 30 s at -20 "C before the reaction was quenched in liquid nitrogen. These spectra were recorded with €b at setting B (free proton frequency (Y,, - x low3, 1.0 x loe3, and 2.0 x M, respectively. In the third spectrum 14.3 MHz). Concentrations of enzyme, substrate, and inhibitor were 1.5

from the top, the feature a t the free proton frequency probably arises through nonspecific binding of the substrate to surface residues. A similar observation was made in the ENDOR study of a-chymotrypsin (18).

ever, the shape of the resonance features was reproducibly and distinguishably different from that of the free substrate or of the product (cf. also Fig. 2).

On this basis, we consider that the resonance features of the reaction intermediate in the matrix region of the spectrum constitute a diagnostic signature of a change in chemical bond- ing structure of the moiety attached to the carboxylate oxygen of the spin-labeled propenoic acid. The resonance features can come only from an amino acid residue of the protein attached to the carboxylic acid group of the propenoyl side chain since the resonance features would not differ substantially from those of the free substrate if an intact TEPOPL molecule were bound in the active site. Also, since the carboxylic acid proton of spin- labeled propenoic acid and the hydroxyl proton of P-L-phenyllactate would exchange with D20, the resonance features cannot be attributed to a tightly bound biproduct-enzyme complex caught after cleavage of the scissile bond. Since the spectrum cannot be accounted for by an intact substrate or by a tightly

I 0.4 MHz Y t

FIG. 4. Proton ENDOR spectra of TEPOPL with & at setting B. The top spectrum is for the substrate (S) free in solution, and the bottom spectrum is for the reaction intermediate ( I ) formed with Ac-CPA. Con- centrations of TEPOPL and Ac-CPA were 1.30 x and 1.65 x M, respectively. The line splittings for H-2 and H-3 in the substrate and in the reaction intermediate are indicated in the stick diagram by solid and dashed lines, respectively. For comparison of line splittings of the free substrate and of the reaction intermediate, peaks at the extreme leR of the diagram assigned to the H-211 component have been aligned. A prominent pair of resonance features (indicated by arrows) is observed in the spectrum of the intermediate of Ac-CPA, and these features are assigned to acetyl protons.

bound biproduct complex and the ENDOR features of the reaction intermediate in the free matrix region are consistent with a mixed-anhydride species formed with the side chain of Glu-270, we assign the spectrum to this acylenzyme reaction intermediate. This interpretation of the nature of the reaction intermediate has been made previously from this laboratory to account for its kinetic and spectroscopic characteristics (1,2,5, 6, 17). Furthermore, it has been demonstrated by nucleophilic trapping studies that the reaction intermediate of CPA formed with a variety of peptide and ester substrates is indeed a mixed-anhydride acylenzyme species (3, 4).

ENDOR of Reaction Intermediate of Ac-CPA Formed with TEPOPGFig. 4 illustrates the ENDOR spectra of the substrate and of the reaction intermediate of TEPOPL formed with Ac-CPA. In Fig. 4, the two resonance features at the extreme left are aligned so that differences in line splittings for H-2 and H-3 in the substrate and intermediate can be more readily recognized. The spectral features of the reaction intermediate of Ac-CPA near the free proton frequency are identical to those of CPA illustrated in Fig. 3. Moreover, a new pair of prominent resonance features (indicated by arrows in Fig. 4) is observed and is assigned to the acetyl protons introduced by chemical modification of active-site tyrosyl residues.

Fig. 5 compares the ENDOR spectra of the reaction intermediates of CPA, Ac-CPA, and [2H31Ac-CPA formed with [3-2H]TE- POPL at -20 "C in 4 M NaCl under identical conditions. The spectrum of the intermediate formed with CPA is essentially identical to the spectrum of the intermediate formed with ['H~IAc-CPA. Since deuteron resonances do not appear in this frequency region, the spectra in both cases arise only from substrate and enzyme protons in the near vicinity of the nitroxyl group. The identical spectra for both reaction intermediates thus indicate that the active-site structure of r2H3]Ac- CPA is isomorphous to that of CPA. This conclusion is further supported by the nearly identical values of KM for CPA and Ac-CPA as shown in Table I.

The resonance features for H-2 are identical in all three


I I I I I

MHz FIG. 5. Proton ENDOR spectra of reaction intermediates of

[3-aH]TEPOPL formed with CPA (fop spectrum), Ac-CPA (middk spectrum), and [sI,IAc-CPA (hottom spectrum). The line splittings for H-2 are indicated by the stick diagrams, and the resonance features for acetyl protons are indicated by arrows. Conditions are the same as described in the legends to Figs. 2-4.

-0.6 -0.3 0 0.3 0.6

spectra. The new resonance features (indicated by arrows in Fig. 5) that are observed only in the spectrum of the reaction intermediate of Ac-CPA and are not seen in the spectrum of the reaction intermediate formed with CPA or [2H,]Ac-CPA are assigned to the acetyl protons. The ENDOR splitting of 0.45 MHz for this line pair is assigned to the perpendicular hyperfine coupling component because it is observed in both magnetic field settings (Ho at settings A and B). The value of this hyperfine coupling tomponent yields an averaged electron-proton distance of 5.6 A. We assign these resonances to the acetyl group attached to Tyr-198 on the basis of molecular modeling, as discussed below.

Molecular Modeling Studies: Conformation of TEPOPL in Active Site of CPA

To determine the conformation of TEPOPL both free in solution and bound in the active site of CPA, we have carried out a computational analysis on the basis of torsion angle search calculations, constrained by ENDOR-determined electron-proton separations listed in Table 11, as described in earlier pub- lications (22-26, 31). Calculations for TEPOPL free in solution with distance constraints to H-2 and H-3 showed that the C(3)=C(2) double bond is essentially coplanar with the oxypyrrolinyl ring of the spin-label. Moreover, the results showed the olefinic bond of the propenoyl side chain to have a planar trans conformation with respect to the olefinic bond in the oxypyrrolinyl ring as measured by the C(4')=C(3')-C(3)=C(2) dihedral angle. On the other hand, torsion angle search calculations constrained by the distances to H-2 and H-3 for the acyl moiety bound in the active site of CPA resulted in a structure of the propenoyl group that deviated significantly from planarity. While the ENDOR-determined electron-proton distances constrained the C(4')=C(3')-C(3)=C(2) and C(3')-C(3)=C(2bC(l) dihedral angles to values of -168" 5 5" and 160" 5 5" in the free substrate, they were -156" 2 10" and 140" 2 10" in the reaction intermediate, respectively. Thus, the main structural difference for the acyl moiety in the free substrate and in the mixed- anhydride reaction intermediate is a torsional change in the

C(3')-C(3)=C(2)-C(l) dihedral angle. In earlier EPR studies of the reaction intermediate formed with TEPOPL, we have also pointed out that accommodation of the spin-labeled propenoyl moiety in the mixed-anhydride species results in torsional dis- tortion and loss of planarity of the propenoyl side chain (17).

Fig. 6 illustrates the acyl moiety of TEPOPL in the active site of CPA as modeled on the basis of the atomic coordinates of the enzyme-inhibitor complex with glycyl-L-tyrosine refined at 1.6-b; resolution (10, 11). The phenolic group of Tyr-248 is shown in the two positions that have been noted for CPA "up" for the free enzyme and "down" for the enzyme-inhibitor complex (7, 10, 11, 36, 37). Only the acyl moiety of the spin-label substrate is shown in the active site. The carbonyl group of the propenoyl side chain has been covalently attached to the y-carboxylate group of Glu-270 to form a mixed-anhydride linkage, with the carbonyl oxygen of the scissile bond coordinated to the Zn2+ ion. The conformation of the substrate is defined by (i) the ENDOR-deErmined distances to H-2 and H-3 and (ii) the distance of 7.8 A between the metal center and the nitroxyl group determined previously in EPR studies of this spin-label substrate with Co2+-reconstituted CPA (17). In this model, the electron-proton distances for the averaged positions of HY'.', HP1s2, and Ha of the Glu-270 side chain range from 8.26 to 9.34 A. As we have shown for the ENDOR spectrum of the intermediate in Fig. 3, the resonance features of these distant but strongly coypled protons yield electron-proton distances of -8.6 2 0.6 A. Both ENDOR and modeling studies are therefore consistent with a mixed-anhydride intermediate. As seen in Fig. 6, the spin-label acyl moiety in its torsionally distorted conformation is nicely accommodated by the calculated solvent- accessible surface (52) of active-site residues.

In Fig. 7 is illustrated a stereodiagram of the acyl moiety of the substrate in the active site of Ac-CPA. The acyl moiety of the substrate is covalently joined to Glu-270. The Zn2+ ion and 2 active-site tyrosine residues in their acetylated forms are also shown with the phenolic ring of "248 in its down position, as generally anticipated in CPA-inhibitor complexes (10, 11). The acetyl group attached to the side chain of 5r-198, as defined in the inhibitor complex with glycyl-L-tyrosine (111, can account exactly for the distance of 5.6 A of the acetyl protons to the nitroxyl group. It is seen that the side chain of Tyr-248 cannot be accommodated in its down position because it overlaps sterically with the spin-labeled propenoyl group and with the phenyllactate moiety in the hydrophobic p ~ c k e t . ~

From this study, we must conclude that the side chain of Tyr-248 cannot be in the same position as in the inhibitor complex with glycyl-L-tyrosine (11) and that it is probably in a position intermediate between those defined as up and down. In an intermediate position, the acetyl protons of Tyr-248 for Ac-CPA would be -10 A distant from the nitroxyl group, with resultant weak hyperfine coupling with the paramagnetic nitroxyl group. In general, it has been observed that the position of the side chain of Tyr-248 is variable, dependent upon the nature of the bound inhibitor ligand (7, 10). In contrast, site- directed mutagenesis studies have shown that while %-248 need not function as a catalytically required residue, the side chain of Tyr-198 may have a role in sterically stabilizing the transition state structure of the bound substrate within the active site (53). This conclusion is consistent with our identification of the position of the side chain of Tyr-198 in van der Waals contact with the acyl moiety of the substrate as shown in Fig. 7. Thus, our identification of the relative positions of W-

It is probable that the phenyllactate group is not released from the SI' binding pocket in the mixed-anhydride reaction intermediate of the enzyme because its release would be sterically blocked by the acyl moiety of the substrate (1, 3, 17).

~~ ~

Catalytic Conformation of Carboxypeptidase A 4593 -

FIG. 6. Stemodhgmm of conformation of the acyl moiety of the win-label substrate in the acylenzyme intermediate of CPA. Catalytically important residues in the active site are labeled, and the Zn- ion is represented by the blue sphere of 2.0-A radius to reflect the average distance of donor-ligand atoms to the metal ion. The side chain of Tyr-248 is shown in t w o positions: the up position described for the free enzyme and the down position as found in the inhibitor complex with glycyl-L-tyrosine. The solvent-accessible surface (52) is represented by dots. The spin-label substrate is shown here covalently bonded to Glu-270, and its conformation is defined by EPR- and ENDOR-determined distances as deauibed in the text. The residues to define the active-site surface were selected by direct examination as described earlier (17).

FIG. 7. Stereodiegram of the acyl moiety of the spin-label substrate in the active site of Ac-CPA. The active-site structure of CPA is based on the x-ray crystallographic studies of the enzyme-inhibitor complex with glycyl-L-tyrosine (11). The acyl moiety of the substrate is covalently bonded to the side chain of Glu-270. The Zn2+ ion (as represented by a sphere), acetyl-Tyr-248 (in its down position), and acetyl-Tyr-198 are shown. The expanded van der Waals surface of the spin-label moiety (shown in blue) and of the phenyllactic acid product (shown in red) is calculated as the solvent-accessible surface (52).

198 and Tyr-248 underlines how application of ENDOR can be critically employed for determination of catalytically important structural relationships.

Stereoelectronic Effects in Hydrolysis of Substrates Catalyzed by Carboxypeptidase A

The results of this ENDOR study to define the conformation of the acyl moiety of the substrate in the active site, when viewed with respect to the structure of the substrate free in solution (26), place important constraints on the structural requirements for catalytic action within the active site of the enzyme. In Fig. 8, we have schematically illustrated the struc-

ture of bound TEPOPL in the active site, the structure of the substrate in which the carbonyl carbon of the scissile bond has sp3 hybridization as in a tetrahedral adduct, and the ENDOR- defined structure of the spin-labeled propenoyl moiety in its mixed-anhydride linkage to Glu-270. For molecular modeling of TEPOPL into the x-ray-defined active site of either CPA (36,371 or the enzyme in the inhibitor complex with glycyl-ttyrosine complex (11). steric accommodation of the intact substrate re- stricted the C(2)-C(1)"Ca dihedral angle to a value of -136", similar to that observed for the spin-labeled ethyl propenoate ester derivative free in solution (26). Furthermore, modeling of the spin-labeled propenoyl moiety to form the mixed-anhydride


FIG. 8. Stereodiagram of the conformation of TEPOPL and of the spin-labeled propemoyl moiety in the active site of CPA. In the upper diagram, the structure of intact bound TEPOPL (solid bonds) is compared to that of the tetrahedral adduct (open bonds). Also illustrated in this diagram is the Glu-270 residue (open bonds). The broken lines connect the tetrahedral carbon to the incoming nucleophilic OE2 of Glu-270. Comparison of the relative positions of the phenyllactate side chain shows that retention of the side chain in a position similar to that of the intact substrate, upon formation of the tetrahedral adduct, re- quires torsional rotation around the ester C-0 bond as well as around O-C", C"-Ca, and CB-CY bonds. The bottom diagram illustrates the spin-labeledpropenoyl moiety in mixed-anhydride linkage with the side chain of Glu-270. In both diagrams, the Z n 2 + ion is shown as a dotted sphere. By careful inspection of the propenoyl side chain, the change in conformation is evident from that of the free substrate to that in the acylenzyme species in which the propenoyl side chain is torsionally altered.

reaction intermediate required the ENDOR-defined conformation of the propenoyl side chain for steric accommodation by active-site residues.

It is seen in Fig. 8 that with nucleophilic attack of a Glu-270 carboxylic oxygen to form a tetrahedral adduct, the equivalent of torsional rotation of the ester C(U-0 bond is required to keep the benzyl side chain within the steric constraints of the hydrophobic SI' binding pocket similar to that seen for the ground state conformation of the bound substrate or that of the tyrosine side chain of the bound glycyl-L-tyrosine inhibitor (10, 11). We have previously described the pathway by which the tetrahedral adduct is cleaved according to stereoelectronic rules to form the mixed-anhydride reaction intermediate (7, 17). The torsional rotation required to produce the tetrahedral adduct for the intact bound substrate in Fig. 8 would occur precisely in the direction for breakdown of the tetrahedral adduct into the mixed-anhydride acylenzyme species according to stereoelectronic rules (54, 55).

The comparison of the structures of the free substrate (26) and of the mixed-anhydride reaction intermediate, together with the x-ray-defined structure of the enzyme active site (8, 10, 11,36,37), yields no alternative set of torsional bond rota-

tions for formation of the tetrahedral adduct and its collapse into a mixed-anhydride reaction intermediate. We have pointed out that in the hydrolysis of esters catalyzed by CPA, kinetically fast isomerization or torsional alteration of the substrate is compatible with the mechanism in which the rate-limiting step is breakdown of the acylenzyme intermediate (7,17). This torsional change in substrate structure upon binding is probably induced through the large amplitude dynamical fluctuations of enzyme residues in subsites Sz and S3 (cf: Fig. 1) since the catalytically required residue Glu-270, the ligands to the Zn2+ ion, and the residues constituting the specificity binding pocket SI' exhibit only low amplitude fluctuations (56). Since the Sz-Pz interactions are responsible for the magnitude of the catalytic rate constant kat (44,451, the modeling studies in this investigation, as constrained by ENDOR-defined electron-proton distances, provide a structural basis for specifying how protein dynamic fluctuations can lead to torsional changes in substrate structure and bond cleavage.

REFERENCES 1. Makinen, M. W., Kuo, L. C., Dymowski, J. J., and JafTer, S. (1979) J. Biol.

2. Makinen. M. W., Fukuyama, J. M., and Kuo, L. C. (1982) J. Am. Chem. Soc.

3. Sander, M. E., and Witzel. H. (1985) Biochem. Bwohvs. Res. Commun. 132.

Chem. 264,356-366

104,2667-2669

4

5. 6. 7.

8. 9.

10.

11.

12.

13.

14. 15.

16. 17.

18.

19. 20. 21. 22. 23.

24.

25.

26.

27. 28. 29.

30. 31.

32.

33.

34. 35. 36. 37.

38.

39. 40.

41.

681-687 Sander, M. E., and Witzel, H. (1986) in Zinc Enzymes (Bertini, I., Luchinat, C.,

Maret, W., and Zeppezauer, M., eds) pp. 207-214, Birkhiiuser Press. Basel, Switzerland

_ -

Kuo, L. C., and Makinen, M. W. (1982) J. Bwl. Chem. 267,24-27

Makinen, M. W., Wells, G. B., and Kang, S. 0. (1984)Adu. Znorg. Biochem. 6, Kuo, L. C., and Makinen, M. W. (1985) J. Am. Chem. Soc. 107,5255-5261

Quiocho, F. A., and Lipscomb, W. N. (1971)Adu. Protein Chem. 25, 1-78 Lipscomb. W. N. (1974) Zktrahedron 30,1725-1732 Rees, D. C., and Lipscomb, W. N. (1981) Proc. Natl. Acad. Sei. U. S. A. 78,

Christianson, D. W., and Lipscomb, W. N. (1986) P m . Natl. Acad. Sci. U. S. A.

Breslow, R., and Wemick, D. L. (1977) P m . Natl. A d . Sei. U. S. A. 74,

Vallee, B. L., and Galdes, A. (1984) M u . Enzymol. Relat. Areas Mol. Biol. 66,

Galdes, A,, Auld, D. S., and Vallee, B. L. (1983) Biochemistry 22, 1888-1893 Geoghegan, K. F., Galdes, A,, Martinelli, R. A,, Hohnquist, B., Auld, D. S., and

Mock, W. L. (1976) Bioorg. Chem. 6,403414 Kuo, L. C., Fukuyama. J. M., and Makinen, M. W. (1983) J. Mol. Biol. 163,

Wells, G. B., Mustafi, D., and Makinen, M. W. (1994) J. Biol. Chem. 289,

Makinen. M. W., and Fink,k L. (1977)Annu. Rev. Bwphys. Biwng. 6,301-343 Douzou, P. (1977) Cryobiochemistry, pp. 286, Academic Press, New York

Cartwright, S. J., and Fink, A. L. (1981) CRC Crit. Reu. Biochem. 11,145-207 Wells, G. B., and Makinen, M. W. (1988) J. Am. Chem. Soc. 110.63434352 Mustafi, D., Sachleben, J. R., Wells, G. B., and Makinen, M. W. (1990) J. Am.

1-69

5455-5459

m, 7568-7572

1303-1307

284430

Vallee, B. L. (1983) Biochemistry 22,2255-2262

63-105

45774586

Wells, G. B., Must&. D., and Makinen, M. W. (1990) J. Am. Chem. Soc. 112, Chem. Soc. 112,2558-2566

2566-2574 Joela, H., Mustafi, D., Fair, C. C., and Makinen, M. W. (1991) J. Phys. Chem.

Mustafi, D., Boisvert, W. E., and Makinen, M. W. (1990) Biopolymers 29,

Simpson, R. T., Riordan, J. F., and Vallee, B. L. (1963) Biochemistry 2,616622 Riordan, J. F., and Vallee, B. L. (1963) Biochemistry 2, 1460-1468 Rozantsev. E. G. (1970) Free Nitroxyl Radicals, pp. 203-246, Plenum Publish-

Suh, J., and Kaiser, E. L. (1976) J. Am. Chem. Soc. 96, 1940-1947 Mustafi, D., Boisvert, W. E., and Makinen, M. W. (1993) J. Am. Chem. Soc. 116,

Koch, T. R., Kuo, L. C., Douglas, E. G., Jaffer, S., and Makinen, M. W. (1979)

Cox, D. J., Bovard, F. C., Bargetzi, J. P., Walsh, K. A,, and Neurath. H. (1964)

Turley, J. W., and Boer, F. P. (1972)Acta Crystallogr Sect. B 28, 164-1644 Cesario, M., and Guilhem, J. (1975) Cryst. S t r u t . Commun. 4,245-248

Rees, D. C.. Lewis, M., Honzatko, R. B., Lipscomb, W. N., and Hardman, K. D. Rees, D. C., Lewis, M., and Lipscomb, W. N. (1983) J. Mol. Biol. 168,367-387

Chian, G., Fmnczek, F. R., Davis, S. T., and Gandour, R. D. (1981) Acta

Jones, T. A. (1985) Methods Enzymol. 116, 157-171 Dayringer, H. E., Tramontano. A., Sprang, S. R., and Fletterick, R. J. (1986) J.

06, 9135-9144

45-55

ing Corp., New York

36743682

J. Bid . Chem. 264,12310-12313

Biochemistry 3 . 4 4 4 7

(1981) P m . Natl. Acad. Sci. U. S. A 78,34083412

Crystallogr Sect. B 37, 1623-1625

Mol.-Gmphics 4, 8 2 4 7 "

Naruto, S., M o b , J., Marshall, G. R., Daniels, S. B., Sofia, M. J., and Kat


42. Iijima, H., Dunbar, J. B., Jr., and Marshall, G. R. (1987) Proteins Struct. Funct.

43. Glovsky, J., Hall, P. L., and Kaiser, E. T. (1972) Biochem. Biophys. Res. Com-

44. Abramowitz, N., Schechter, I., and Berger, A. (1967) Biochem. Bwphys. Res.

45. Schechter, I. (1970) E m J. Biochem. 14,516-520 46. Griffith, 0. H., Cornell, D. W., and McConnell, H. M. (1965) J. Chem. Phys. 43,

47. Capiomont, A,, Chion, B., Lajzerowich-Bonneteau, J., and Lemaire, H. (1974)

48. Berliner, L. J. (ed) (1976) Spin Label ing: Theory and Application, pp. 592,

zenellenbogen, J. A. (1985) J. Am. Chem. Soc. 107,52624270

Genet. 2,330-339

mun. 47,244-247

Commun. 29,862-867

2909-2910

J. Chem. Phys. 60,2530-2535

49. Mustafi, D., Joela, H., andMakinen, M. W. (1991)J. Magn. Reson. 91,497-504 50. Lumry, R., Smith, E. L., and Glantz, R. R. (1951) J. Am. Chem. Soc. 73,

51. Byers, L. D., and Wolfenden, R. (1973) Biochemistry 12,2070-2078 52. Lee, B., and Richards, F. M. (1971) J. Mol. Biol. 56, 379-400 53. Gardell, S. J., Hilvert, D., Burnett, J., Kaiser, E. T., and Rutter, W. J. (1987) J.

54. Deslongchamps, P. (1975) Zbtmhedron 31,2463-2490 55. Deslongchamps, P. (1983) Stereoelectronic Eflects in Organic Chemistry, pp.

56. Makinen, M. W., Troyer, J. M., van der WerfT, H., Berendsen, H. J. C., and van

Academic Press, New York

4330-4340

Biol. Chem. 262,576-582

54-162, Pergamon Press, New York

Gunsteren, W. F. (1989) J. Mol. Biol. 207,201-216

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Catalytic Conformation of Carboxypeptidase A