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PubMed2001

Binding of the glutamate carboxypeptidase II (NAALADase) inhibitor 2-PMPA to rat brain membranes.

2-Phosphonomethyl pentanedioic acid (2-PMPA) is a potent and selective inhibitor of glutamate carboxypeptidase II (NAALADase), and has shown robust neuroprotective activity in both in vitro and in vivo models of ischemia. In the brain, glutamate carboxypeptidase II (GCPII) (EC3.4.17.21) hydrolyzes the neuropeptide N-acetylaspartylglutamate (NAAG) to glutamate and N-acetylaspartate. We report the development and characterization of a [(3)H]2-PMPA binding assay. [(3)H]2-PMPA binding was dependent on protein concentration, saturable, and displaceable. The association (k(on)) and dissociation (k(off)) rate constants were 3x10(6) M(-1) s(-1) and 0.01 s(-1), respectively. The dissociation equilibrium constant (K(d)) determined from the ratio of the rate constants (K(d)=k(off)/k(on)) was 1 nM. Scatchard analysis revealed one binding site with K(d)=2 nM and B(max)=0.7 pmol/mg. Binding exhibited similar pharmacological properties to GCPII enzyme activity, including chloride dependency, cobalt stimulation and inhibition by phosphate and quisqualate. The binding of [(3)H]2-PMPA also showed tissue specificity in that tissues previously reported to be devoid of GCPII enzymatic activity were devoid of [(3)H]2-PMPA binding. [(3)H]2-PMPA binding represents an additional probe for the study of GCPII activity, and may be useful as a high throughput screening assay.

Animals↗

PubMed1998

Production and characterization of monoclonal anti-idiotypic antibody exhibiting a catalytic activity similar to carboxypeptidase A.

A new approach for producing catalytic anti-idiotypic antibody was developed. A monoclonal anti-idiotypic antibody which was induced against carboxypeptidase A (CPA) showed the catalytic activity similar to the original antigen. The activity of the catalytic antibody was investigated. Rabbits were immunized by bovine pancreas carboxypeptidase A. The antiserum was purified and used as antigen to immunize BALB/c mice to induce monoclonal anti-idiotypic antibodies. Screened for enzymatic activities, the monoclonal antibody 32C3 showed esterase activity. The hydrolysis of hippuryl-DL-phenyllactic acid by McAb 32C3 followed the enzymatic kinetics. In our experimental system, Kcat value was 0.0123 min-1 and Km was 0.04M. The acceleration rate was 1750 times compared to the rate of self-hydrolysis of the substrate. This hydrolysis reaction can be competitively inhibited by hydrocinnamic acid. This method could be effective to obtain catalytic antibodies with the characters close to natural enzymes.

Animals↗

PubMed2002

Syntheses and kinetic evaluation of racemic and optically active 2-benzyl-2-methyl-3,4-epoxybutanoic acids as irreversible inactivators for carboxypeptidase A.

Racemic and optically active 2-benzyl-2-methyl-3,4-epoxybutanoic acids were synthesized and evaluated as inactivators for carboxypeptidase A, a representative zinc-containing proteolytic enzyme. Only the threo-form of the inactivator is effective and its potency in terms of k(inact)/K(I) value is lower by 42-fold compared with 2-benzyl-3,4-epoxybutanoic acid, indicating that the alpha-methyl group affects adversely in the inactivation contrary to the expectation that it would enhance the inactivation activity of the inhibitor through additional interactions of the methyl group with a small cavity (alpha-methyl hole) present next to the S1' hydrophobic pocket. Of the enantiomeric pair, the inactivator having the (2S,3R)-configuration is more potent than its enantiomer by 44-fold. The observed kinetic results may be rationalized on the basis that the methyl group in the inactivator having the (2R,3S)-configuration experiences the van der Waals repulsive interactions with the bottom of the active site crevice in binding to CPA, casting a doubt on the presence of the so-called alpha-methyl hole at the active site of carboxypeptidase A.

Animals↗

PubMed2002

Isolation and characteristics of carboxypeptidase B from the pyloric ceca of the starfish Asterias amurensis.

Carboxypeptidase B was purified from the pyloric ceca of the starfish Asterias amurensis. The final enzyme preparation was nearly homogeneous in polyacrylamide gel electrophoresis and its molecular weight was estimated as approximately 34,000. The optimum pH and temperature of the enzyme for hydrolysis of benzoyl-glycyl-L-arginine were at approximately pH 7.5 and 55 degrees C, respectively. The enzyme was unstable at above 50 degrees C and at below pH 5.0. The enzyme was activated by Co(2+), but was inhibited by EDTA and Hg(2+). The N-terminal amino acid sequence of A. amurensis carboxypeptidase B was ASFDYNVYHSYQEIMNWITN.

Amino Acid Sequence↗

PubMed2000

Inhibition of carboxypeptidase A by D-penicillamine: mechanism and implications for drug design.

Zinc metalloprotease inhibitors are usually designed to inactivate the enzyme by forming a stable ternary complex with the enzyme and active-site zinc. D-Cysteine inhibits carboxypeptidase, ZnCPD, by forming such a complex, with a K(i) of 2.3 microM. In contrast, the antiarthritis drug D-penicillamine, D-PEN, which differs from D-Cys only by the presence of two methyl groups on the beta-carbon, inhibits ZnCPD by promoting the release of the active-site zinc. We have given the name catalytic chelator to such inhibitors. Inhibition is a two-step process characterized by formation of a complex with the enzyme (K(i(initial)) = 1.2 mM) followed by release of the active-site zinc at rates up to 420-fold faster than the spontaneous release. The initial rate of substrate hydrolysis at completion of the second step also depends on D-PEN concentration, reflecting formation of a thermodynamic equilibrium governed by the stability constants of chelator and apocarboxypeptidase for zinc (K(i(final)) = 0.25 mM). The interaction of D-PEN and D-Cys with the active-site metal has been examined by replacing the active-site zinc by a chromophoric cobalt atom. Both inhibitors perturb the d-d transitions of CoCPD in the 500-600 nm region within milliseconds of mixing but only the CoCPD.D-Cys complex displays a strong S --> Co(II) charge-transfer band at 340 nm indicative of a metal-sulfur bond. While the D-Cys complex is stable, the CoCPD.D-PEN complex breaks down to apoenzyme and Co(D-PEN)(2) with a half-life of 0.5 s. D-PEN is the first drug found to inhibit a metalloprotease by increasing the dissociation rate constant of the active-site metal. The ability of D-PEN to catalyze metal removal from carboxypeptidase A and other zinc proteases suggests a possible mechanism of action in arthritis and Wilson's disease and may also underlie complications associated with its clinical use.

Carboxypeptidases↗

PubMed1992

Activation of methotrexate-alpha-alanine by carboxypeptidase A-monoclonal antibody conjugate.

Carboxypeptidase A (CP-A) and monoclonal antibody KS1/4 directed against an antigen on human lung adenocarcinoma cells (UCLA-P3) were derivatized by treatment with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and N-succinimidyl 3-(2-pyridyldithio)propionate, respectively. The derivatized proteins were reacted to produce thioether-linked enzyme-antibody conjugates. Sequential HPLC size-exclusion and DEAE chromatography separated the conjugate preparation from unreacted enzyme and antibody. On the basis of SDS-PAGE analysis and measurement of catalytic activity, the preparation contained approximately equal amounts of 1:1 and 2:1 (enzyme:antibody) conjugates; binding activity of the conjugate (1.8 x 10(5) molecules/cell) was similar to that of unreacted antibody. In vitro cytotoxicity studies with UCLA-P3 cells demonstrated the ability of cell-bound conjugate to convert the prodrug methotrexate-alpha-alanine (MTX-Ala) to methotrexate (MTX). In the absence of conjugate, ID50 values for MTX-Ala and MTX were 8.9 x 10(-6) and 5.2 x 10(-8) M, respectively. ID50 for the prodrug improved to 1.5 x 10(-6) M with cells containing bound conjugate. This potentiation of MTX-Ala cytotoxicity by conjugate-bound CP-A, which was at least 30-fold greater than that produced by a comparable amount of free enzyme, is attributed to enhanced effectiveness of MTX generated at the cell surface as opposed to the surrounding medium. Examination of the time course of cytotoxicity over a 96-h period showed that the conjugate-prodrug combination (at 2.5 x 10(-6) M) was nearly as effective as MTX in preventing cell replication. These results demonstrate the chemotherapeutic potential of carboxypeptidase-monoclonal antibody conjugates used in conjunction with MTX peptide prodrugs.

Adenocarcinoma↗

PubMed1992

pH-dependent properties of cobalt(II) carboxypeptidase A-inhibitor complexes.

1H NMR spectroscopy of the isotropically shifted signals in cobalt carboxypeptidase, CoCPD, permits a direct and selective detection of protons belonging to the residues liganded to the metal. The chemical shift of these protons in the free enzyme and enzyme-inhibitor complexes with changing pH monitors the state of ionization of the ligands directly and of other residues in the active center indirectly. The 1H NMR spectrum of CoCPD at pH 6 shows three well-resolved isotropically shifted signals in the downfield region at 62 (a), 52 (c), and 45 (d) ppm which have been assigned to the NH proton of His-69 and to the C-4 H's of His-69 and His-196, respectively. Titration of signal a with pH is characterized by a pKa of 8.8 which is identical to that seen in prior electronic absorption and kinetic studies. The fact that the signal reflecting the NH of His-69 is still observed at pH 10 and no major shifts occur for the signals reflecting the C-4 H's indicates the alkaline pKa in carboxypeptidase A catalysis, pKEH, cannot be ascribed to ionization of the histidyl NH of either His-69 or His-196. Binding of L-Phe shifts this pKa to 7.7 while not greatly perturbing the downfield 1H NMR signals that reflect the ligation shell of the cobalt coordination sphere. These results indicate the pKa of 8.8 in CoCPD and the pKa of 7.7 in the CoCPD.L-Phe adduct reflect ionization of the same group.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2000

Principles of hydroxamate inhibition of metalloproteases: carboxypeptidase A.

Hydroxamic acids of structure RCON(OH)CH(2)CH(CH(2)C(6)H(5))CO(2)H induce micromolar competitive inhibition of catalysis for the enzyme carboxypeptidase A. Enzyme affinity depends on the nature of the acyl group, for RCO equaling HCO, CH(3)CO, FCH(2)CO, F(2)CHCO, F(3)CCO, CH(3)OCH(2)CO, or CH(3)OCO. In acid dissociation these residues yield hydroxamic acid pK(a) values that vary from 7.6 to 10.3. Profiles of inhibitory pK(i) plotted versus pH indicate characteristically a maximum effectiveness near neutrality. Weaker binding to enzyme is generally displayed in either acidic or alkaline solution, with the position of the alkaline limb of the profiles depending on the pK(a) of the inhibitor. A reverse-protonation pattern of association with the enzyme is indicated, in which the hydroxamate anion of the inhibitor displaces a relatively acidic H(2)O ligand (pK(a) of 6) from the active-site zinc ion of carboxypeptidase A. The metal-coordinating, N-substituted hydroxamic acid functional groups exist in solution as a mixture of syn and anti rotamers, with relative abundances that depend on their pK(a). A pyrrolidinone analogue having a conformationally syn-fixed cyclohydroxamic acid was not an especially potent inhibitor. Structure-activity relationships suggest design criteria for hydroxamic acid inhibitors in order to provide most effective binding with metalloenzymes.

Animals↗

PubMed1991

Characterization of an inhibitory metal binding site in carboxypeptidase A.

The specificity of metal ion inhibition of bovine carboxypeptidase A ([(CPD)Zn]) catalysis is examined under stopped-flow conditions with use of the fluorescent peptide substrate Dns-Gly-Ala-Phe. The enzyme is inhibited competitively by Zn(II), Pb(II), and Cd(II) with apparent KI values of 2.4 x 10(-5), 4.8 x 10(-5), and 1.1 x 10(-2) M in 0.5 M NaCl at pH 7.5 and 25 degrees C. The kcat/Km value, 7.3 x 10(6) M-1 s-1, is affected less than 10% at 1 x 10(-4) M Mn(II) or Cu(II) and at 1 x 10(-2) M Co(II), Ni(II), Hg(II), or Pt(IV). Zn(II) and Pb(II) are mutually exclusive inhibitors. Previous studies of the pH dependence of Zn(II) inhibition [Larsen, K. S., & Auld, D. S. (1989) Biochemistry 28, 9620] indicated that [(CPD)Zn] is selectively inhibited by a zinc monohydroxide complex, ZnOH+, and that ionization of a ligand, LH, in the enzyme's inhibitory site (pKLH 5.8) is obligatory for its binding. The present study allows further definition of this inhibitory zinc site. The ionizable ligand (LH) is assigned to Glu-270, since specific chemical modification of this residue decreases the binding affinity of [(CPD)Zn] for Zn(II) and Pb(II) by more than 60- and 200-fold, respectively. A bridging interaction between the Glu-270-coordinated metal hydroxide and the catalytic metal ion is implicated from the ability of Zn(II) and Pb(II) to induce a perturbation in the electronic absorption spectrum of cobalt carboxypeptidase A ([(CPD)Co]).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Synthesis and evaluation of an inhibitor of carboxypeptidase A with a Ki value in the femtomolar range.

Comparative studies among a series of tripeptide phosphonate inhibitors of the zinc peptidase carboxypeptidase A indicate that incorporation of the phosphonic acid analogue of valine at the P1 position results in significantly higher affinity than the glycine, alanine, or phenylalanine analogues. When applied to the tripeptide analogue Cbz-Phe-ValP-(O)Phe [ZFVP(O)F], determination of the inhibition constant Ki was complicated by the very slow rate of dissociation. The rate of exchange of [3H]ZFVP(O)F with enzyme-bound [14C]ZFVP(O)F was followed for periods of 3-4 months to measure dissociation rate constants in the range of (1.7-4.4) x 10(-9) s-1, corresponding to half-lives of 5-13 years. Although the on- and off-rate constants differ for different carboxypeptidase isozymes, their ratios, corresponding to the inhibition constants Ki, are consistently in the range of 10-27 fM. Both the inhibition constants and the dissociation rate constants appear to be the lowest values yet determined for an enzyme-small inhibitor interaction.

Carboxypeptidases↗

PubMed1983

Spectral properties of cobalt carboxypeptidase A. Interaction of the metal atom with anions.

At pH greater than 7 the absorption and magnetic circular dichroic spectra of cobalt carboxypeptidase A are insensitive to anions [Latt, S. A., & Vallee, B. L. (1971) Biochemistry 10, 4263-4270], but at pH less than 6 chloride and other anions perturb them in a manner specific for each anion. Lowering of the pH apparently facilitates the entry of an anion into the metal coordination sphere, suggesting that an acidic group normally stabilizes a metal-coordinated water molecule against displacement. The lack of sensitivity to anions at pHs between 7 and 9--when the enzyme is maximally active--and its evident abolition upon protonation of an active-site group are consistent with this interpretation. Selective modification of cobalt carboxypeptidase at Glu-270 using a carbodiimide affinity reagent generates sensitivity to anions at pH 7 very similar to that of the unmodified enzyme at pH approximately 5. This suggests that the group stabilizing the metal-coordinated water is the catalytically essential carboxylate of Glu-270. These and related results provide evidence for a mechanistically important interaction of Glu-270 with a metal-bound water molecule.

Binding Sites↗

PubMed1984

Inhibition of carboxypeptidase A by aldehyde and ketone substrate analogues.

DL-2-Benzyl-3- formylpropanoic acid ( XIVb ) is a competitive inhibitor of carboxypeptidase A with an apparent Ki of 0.48 microM at pH 7.5 in 50 mM Tris buffer-0.5 M in sodium chloride with O-(trans-p- chlorocinnamoyl )-L-beta-phenyllactate as substrate. At pH 7.5 in deuterium oxide, DL-2-benzyl-3- formylpropanoic acid exists as an equilibrium mixture of 75% free aldehyde and 25% hydrated aldehyde. The species that binds to the enzyme may be either the free aldehyde or the hydrate. Therefore, the Ki of the species bound is significantly less than the observed Ki of 0.48 microM. The alcohol and dioxolane analogues of this aldehyde, DL-2-benzyl-4-hydroxybutanoic acid (XI) and 2-benzyl-4,4-(ethylenedioxy)butanoic acid ( XXVII ), are only weak inhibitors with Ki's of 0.54 mM and 2 mM, respectively. The ketone, (+/-)-3-(p- methoxybenzoyl )-2- benzylpropanoic acid [(+/-)-I; Sugimoto , T., & Kaiser, E. T. (1978) J. Am. Chem. Soc. 100, 7750-7751], was found to have a Ki of 180 microM, experimentally indistinguishable from that of the diastereomeric mixture of its alcohol analogue 2-benzyl-4-hydroxy-4-(p-methoxyphenyl)butanoic acid (III), Ki = 190 microM. The ketone (I) is not detectably hydrated (less than 2%) at pH 7.5 in deuterium oxide. These results suggest that the hydratable aldehyde DL-2-benzyl-3- formylpropanoic acid may mimic an intermediate resembling the transition state for amide hydrolysis by carboxypeptidase A while the nonhydratable ketone does not do so.

Aldehydes↗

PubMed1987

Interaction of carboxypeptidase A with carbamate and carbonate esters.

The carbamate ester N-(phenoxycarbonyl)-L-phenylalanine binds well to carboxypeptidase A in the manner of peptide substrates. The ester exhibits linear competitive inhibition toward carboxypeptidase A catalyzed hydrolysis of the amide hippuryl-L-phenylalanine (Ki = 1.0 X 10(-3) M at pH 7.5) and linear noncompetitive inhibition toward hydrolysis of the specific ester substrate O-hippuryl-L-beta-phenyllactate (Ki = 1.4 X 10(-3) M at pH 7.5). Linear inhibition shows that only one molecule of inhibitor is bound per active site at pH 7.5. The hydrolysis of the carbamate ester is not affected by the presence of 10(-8)-10(-9) M enzyme (the concentrations employed in inhibition experiments), but at an enzyme concentration of 3 X 10(-6) M catalysis can be detected. The value of kcat at 30 degrees C, mu = 0.5 M, and pH 7.45 is 0.25 s-1, and Km is 1.5 X 10(-3) M. The near identity of Km and Ki shows that Km is a dissociation constant. Substrate inhibition can be detected at pH less than 7 but not at pH values above 7, which suggests that a conformational change is occurring near that pH. The analogous carbonate ester O-(phenoxycarbonyl)-L-beta-phenyllactic acid is also a substrate for the enzyme. The Km is pH independent from pH 6.5 to 9 and has the value of 7.6 X 10(-5) M in that pH region. The rate constant kcat is pH independent from pH 8 to 10 at 30 degrees C (mu = 0.5 M) with a limiting value of 1.60 s-1. Modification of the carboxyl group of glutamic acid-270 to the methoxyamide strongly inhibits the hydrolysis of O-(phenoxycarbonyl)-L-beta-phenyllactic acid. Binding of beta-phenyllactate esters and phenylalanine amides must occur in different subsites, but the ratios of kcat and kcat/Km for the structural change from hippuryl to phenoxy in each series are closely similar, which suggests that the rate-determining steps are mechanistically similar.

Carbamates↗

PubMed1989

Localization of a highly immunogenic region of carboxypeptidase A recognized by three different monoclonal antibodies and their use in the detection of subtle conformational alterations in this enzyme region.

Three murine monoclonal antibodies (mAb 100, 104, and 121) elicited against carboxypeptidase A (CPA) were prepared and characterized. All three mAbs recognize the same or partially overlapping sites of CPA. This is corroborated by the lack of antibody additivity in the ELISA assay carried out in the presence of pairs of mAbs, the similarity in molecular weight of the immunocomplex formed between CPA and one of the mAbs in the presence of another, and also a competition experiment in which one of the mAbs was labeled enzymatically. The three mAbs do not affect the enzymatic activity of CPA. Even at high concentrations, they do not recognize carboxypeptidase B (CPB) in spite of the similar tertiary structure and the 50% homology in amino acid sequence with CPA. This antigenic determinant is located on one of the four cyanogen bromide fragments of CPA. On the basis of the known sequences of the two enzymes, criteria which predict high antigenicity, and experimental data using synthetic peptides, such a determinant was found to be located within the amino acid sequence from residues 209 to 218 of the CPA molecule. The mAbs prepared detect conformational alterations in the above enzyme epitope when the enzyme is exposed to various conditions. The binding of the mAbs to CPA adsorbed onto a polystyrene plate is characterized by apparent binding constants higher by 1 or 2 orders of magnitude than those characterizing the interaction of the mAbs with CPA in solution. The mAbs also readily detect both conformational alterations of CPA on treatment with urea and subtle, reversible conformational alterations on removal of zinc from the active site of the enzyme.

Amino Acid Sequence↗

PubMed1989

Characterization of the inhibitor complexes of cobalt carboxypeptidase A by electron paramagnetic resonance spectroscopy.

The metal coordination sphere of cobalt-substituted carboxypeptidase A and its complexes with inhibitors has been characterized by X-band electron paramagnetic resonance (EPR) spectroscopy. The temperature dependence of the EPR spectrum of cobalt carboxypeptidase and the g anisotropy are consistent with a distorted tetrahedral geometry for the cobalt ion. Complexes with L-phenylalanine, a competitive inhibitor of peptide hydrolysis, as well as other hydrophobic L-amino acids all exhibit very similar EPR spectra described by three g values that differ only slightly from that of the cobalt enzyme alone. In contrast, the EPR spectra observed for the cobalt enzyme complexes with 2-(mercaptoacetyl)-D-Phe, L-benzylsuccinate, and L-beta-phenyllactate all indicate an approximately axial symmetry of the cobalt atom in a moderately distorted tetrahedral metal environment. Phenylacetate, beta-phenylpropionate, and indole-3-acetate, which exhibit mixed modes of inhibition, yield EPR spectra indicative of multiple binding modes. The EPR spectrum of the putative 2:1 inhibitor to enzyme complex is more perturbed than that of the 1:1 complex. For beta-phenylpropionate, partially resolved hyperfine coupling (122 x 10(-4) cm-1) is observed on the g = 5.99 resonance, possibly indicating a stronger metal interaction for this binding mode. The structural basis for the observed EPR spectral perturbations is discussed with reference to the existing crystallographic kinetic and electronic absorption, nuclear magnetic resonance, and magnetic circular dichroic data.

Carboxypeptidases↗

PubMed1989

Phosphonate analogues of carboxypeptidase A substrates are potent transition-state analogue inhibitors.

Analogues of tri- and tetrapeptide substrates of carboxypeptidase A in which the scissile peptide linkage is replaced with a phosphonate moiety (-PO2--O-) were synthesized and evaluated as inhibitors of the enzyme. The inhibitors terminated with either L-lactate or L-phenyllactate [designated (O) Ala and (O) Phe, respectively] in the P1' position. Transition-state analogy was shown for a series of 14 tri- and tetrapeptide derivatives containing the structure RCO-AlaP-(O)Ala [RCO-AP(O)A, AP indicates the phosphonic acid analogue of alanine] by the correlation of the Ki values for the inhibitors and the Km/kcat values for the corresponding amide substrates. This correlation supports a transition state for the enzymatic reaction that resembles the tetrahedral intermediate formed upon addition of water to the scissile carbonyl group. The inhibitors containing (O) Phe at the P1' position proved to be the most potent reversible inhibitors of carboxypeptidase A reported to date: the dissociation constants of ZAFP(O)F, ZAAP(O)F, and ZFAP(O)F are 4, 3, and 1 pM, respectively. Because of the high affinity of these inhibitors, their dissociation constants could not be determined by steady-state methods. Instead, the course of the association and dissociation processes was monitored for each inhibitor as its equilibrium with the enzyme was established in both the forward and reverse directions. A phosphonamidate analogue, ZAAPF, in which the peptide linkage is replaced with a -PO2-NH- moiety, was prepared and shown to hydrolyze rapidly at neutral pH (t1/2 = 20 min at pH 7.5). This inhibitor is bound an order of magnitude less tightly than the corresponding phosphonate, ZAAP(O)F, a result that contrasts with the 840-fold higher affinity of phosphonamidates for thermolysin [Bartlett, P. A., & Marlowe, C. K. (1987) Science 235, 569-571], a zinc peptidase with a similar arrangement of active-site catalytic residues.

Animals↗

PubMed1998

Native carboxypeptidase A in a new crystal environment reveals a different conformation of the important tyrosine 248.

Native carboxypeptidase A has been crystallized in a new crystal form, and the structure has been refined with X-ray data to 2.0 A resolution. In contrast to the previously published structure [Rees, D. C., Lewis, M., and Lipscomb, W. N. (1983) J. Mol. Biol. 168, 367-387], no active-site amino acids are involved in the crystal packing. The important Tyr248 is stabilized inside the active site by a hydrogen bond and by interactions with Ile247. The proposed role of Tyr248 in the induced fit mechanism is therefore not supported by the findings in this structure of native carboxypeptidase A. The structure has a partly populated inhibitory Zn2+ site in close proximity to the catalytic Zn2+ as evident from X-ray anomalous dispersion data. A hydroxo bridge is found between the catalytic Zn2+ and the inhibitory Zn2+ with a Zn2+-Zn2+ distance of 3.48 A. In addition, the inhibitory Zn2+ has Glu270 as a monodentate ligand. No other protein ligands to the inhibitory Zn2+ are seen. The crystals were grown at 0.3 M LiCl and weak evidence for a binding site for partly competitive inhibitory anions is observed.

Animals↗

PubMedDate not supplied

Site-directed mutagenesis shows that tyrosine 248 of carboxypeptidase A does not play a crucial role in catalysis.

The residue Tyr 248 of carboxypeptidase A (CPA) is thought to play a role in catalysis by contributing a proton to the incipient amine anion generated during cleavage of peptide substrates. To test this hypothesis we have modified the rat CPA cDNA by site-directed mutagenesis so that the codon for Tyr 248 is replaced by that for Phe. Here, we report the expression of the cDNAs for proCPA and its Tyr-to-Phe variant in yeast via the alpha-factor system. Following zymogen activation by trypsin, wild-type CPA (CPA-WT) and variant CPA (CPA-Phe 248) were purified to homogeneity and characterized enzymatically. CPA-Phe 248 displays essentially undiminished values for the catalytic constant (kcat) towards various peptide and ester substrates. However, the Michaelis constants (Km values) of peptide substrates and the inhibition constant (Ki) of the potato carboxypeptidase inhibitor are increased 6-fold and 70-fold, respectively. These data suggest that the phenolic hydroxyl of Tyr 248 does not act as the requisite general acid catalyst but participates in ligand binding.

Amino Acid Sequence↗