Bovine Pancreatic Carboxypeptidase Research Articles

Carboxypeptidase A-catalyzed direct conversion of leukotriene C4 to leukotriene F4.

Leukotrienes (LTs) are 5-lipoxygenase (5-LO)-derived arachidonic metabolites that constitute a potent set of lipid mediators produced by inflammatory cells. Leukotriene A(4), a labile allylic epoxide formed from arachidonic acid by dual 5-LO activity, is the precursor for LTB(4) and LTC(4) synthesis. LTC(4) is further transformed enzymatically by the sequential action of gamma-glutamyltranspeptidase and dipeptidase to LTD(4) and LTE(4), respectively. In this report, we present evidence that bovine pancreatic carboxypeptidase A (CPA), which shares significant sequence homology with CPA in mast cell granules, catalyzes the conversion of LTC(4) to LTF(4) via the hydrolysis of an amide bond. The identity of CPA-catalyzed LTC(4) hydrolysis product as LTF(4) was confirmed by several analytical criteria, including enzymatic conversion to conjugated tetraene by soybean LO, conversion to LTE(4) by gamma-glutamyltranspeptidase, cochromatography with the standard LTF(4) and positive-ion fast-atom bombardment mass spectral analysis. Thus, it appears that the physiological significance of this single-step transformation may point toward a major cellular homeostatic mechanism of metabolizing LTC(4), a potent bronco- and vasoconstrictor, to a less potent form of cysteinyl LTs.

Expression of a soluble and activatable form of bovine procarboxypeptidase A in Escherichia coli

Bovine pancreatic procarboxypeptidase A has been overexpressed in a soluble and activatable form in Escherichia coli. When the protein was expressed under the control of bacteriophage T7 promoter in E. coli ADA494 (a thioredoxin reductase deficient bacteria), a thioredoxin fusion protein was produced at relatively high level in the cytoplasm (4 mg/L culture medium). Although the recombinant protein essentially accumulated as inclusion bodies, as much as 30% of the fusion protein was recovered in a soluble form at low growth temperature and could therefore be purified to homogeneity in a single-step procedure by metal-affinity chromatography. The recombinant precursor form of bovine carboxypeptidase A was recognized by a monoclonal antibody directed against purified bovine pancreatic carboxypeptidase A. Moreover, upon tryptic activation it gave rise to an enzyme, the N-terminal sequence, molecular size,and specific activity of which were comparable to those of the enzyme derived from the native precursor purified from bovine pancreas.

SYNTHESIS OF METHOTREXATE PRODRUGS AS AN APPROACH FOR DRUG TARGETING

Eleven methotrexale-alpha-peptides containing arginine (MTX-Arg), asparagine (MTX-Asn), cysteine (MTX-Cys), glutamine (MTX-Gln), histidine (MTX-His), lysine (MTX-Lys), methionine (MTX-Met), phenylalanine (MTX-Phe), proline (MTX-Pro), tryptophan (MTX-Trp) and tyrosine (MTX-Tyr) were produced by solid phase synthesis using the Fmoc method. Purity of the MTX derivatives was assessed by capillary zone electrophoresis (CZE). An enzymatic assay was then developed using CZE for monitoring the hydrolysis of the MTX-alpha-peptides by bovine pancreatic carboxypeptidase A (CP-A) leading to the release of free MTX. MTX-Phe appeared to be the most suitable substrate for CP-A out of the eleven tested with a hydrolysis rate comparable to that of hippuryl-L-phenylalanine, a natural substrate for CP-A. In vitro assays on a human ovarian teratocarcinoma cell line (CRL-1572), showed that MTX-Phe was non toxic, but when combined with 1 mU of CP-A, MTX-Phe cytotoxicity was enhanced considerably showing only two times less pharmacological activity than MTX. These results suggest that MTX-Phe is a potent prodrug and could be used in a drug targeting model combining monoclonal antibodies coupled with CP-A for a more specific approach in cancer therapy.

Human Skin Mast Cell Carboxypeptidase: Functional Characterization, cDNA Cloning, and Genealogy

We functionally characterized human skin mast cell carboxypeptidase A (MC-CPA), and explored its evolutionary relationship to other carboxypeptidases to understand further the structural basis for the substrate preferences of this enzyme. Purified human skin MC-CPA displayed more activity than did bovine pancreatic carboxypeptidase A (CPA) against carboxyl-terminal leucine residues, about equal activity with phenylalanine and tyrosine residues, and no activity with tryptophan or alanine. To correlate kinetic data with structure, we isolated and sequenced a cDNA encoding MC-CPA from human skin, and directly sequenced 30% of the purified protein. These sequences agreed with that of human lung MC-CPA, and further support the evidence for a single MC-CPA gene in humans. Four amino acid replacements, resulting in a net positive change in non-hydrogen atoms in the S1' subsite of MC-CPA, were associated with less alteration in substrate specificity, relative to bovine CPA, than might be expected from studies using rat CPA1 and CPA2. We noted two consensus N-linked glycosylation sites in human MC-CPA that are not found in rat and mouse MC-CPA, or in bovine CPA; that at least one of these sites is glycosylated in vivo was verified by N-glycosidase F treatment, lentil lectin binding, and Concanavalin A-Sepharose chromatography. Evolutionary trees constructed from the known carboxypeptidase sequences suggested that MC-CPA most likely evolved from a carboxypeptidase B-like enzyme, independent of the pancreatic CPA. Thus, in the carboxypeptidase gene family, MC-CPA displays a unique genealogy and several amino acid replacements in its S1' binding pocket that result in substrate specificity quite similar to bovine CPA.

Metallotropic forms of zinc, copper and cadmium carbonic anhydrases and zinc carboxypeptidases reflected in differential pulse polarography

Bovine erythrocyte carbonic anhydrase II (ZnCA), its metal substituted analogs CuCA, CdCA and bovine pancreatic carboxypeptidase A (ZnCP) were studied by means of differential pulse polarography (DPP) in buffered media in the pH range of 7.0 to 9.0. Each of the metalloproteins studied yielded several DPP peaks which were attributed to the reduction of several types of metal centre. These centres arose through migration of the single Me(II) to other donor groups (forms B and D) than the groups known from crystals (forms A). The peak potentials of the known structures A of ZnCA and ZnCP allowed insight into the structures B and D. Surface denaturation and the interaction of metallotropic forms of the enzymes with inhibitors are discussed.

Metallotropic forms of zinc, copper and cadmium carbonic anhydrases and zinc carboxypeptidase reflected in differential pulse polarography

Bovine erythrocyte carbonic anhydrase II (ZnCA), its metal substituted analogs CuCA, CdCA and bovine pancreatic carboxypeptidase A (ZnCP) were studied by means of differential pulse polarography (DPP) in buffered media in the pH range of 7.0 to 9.0. Each of the metalloproteins studied yielded several DPP peaks which were attributed to the reduction of several types of metal centre. These centres arose through migration of the single Me(II) to other donor groups (forms B and D) than the groups known from crystals (forms A). The peak potentials of the known structures A of ZnCA and ZnCP allowed insight into the structures B and D. Surface denaturation and the interaction of metallotropic forms of the enzymes with inhibitors are discussed.

Molecular Cloning and Sequencing of the cDNA for Human Membrane-bound Carboxypeptidase M: Comparison with carboxypeptidases A, B, H, and N

Abstract Carboxypeptidase M, a widely distributed membrane-bound carboxypeptidase that can regulate peptide hormone activity, was purified to homogeneity from human placenta (Skidgel, R. A., Davis, R. M., and Tan, F. (1989) J. Biol. Chem. 264, 2236-2241). The NH2-terminal 31 amino acids were sequenced, and two complementary oligonucleotide probes were synthesized and used to isolate a carboxypeptidase M clone from a human placental cDNA library. Sequencing of the cDNA insert (2009 base pairs) revealed an open reading frame of 1317 base pairs coding for a protein of 439 residues. The NH2-terminal protein sequence matched the deduced amino acid sequence starting with residue 14. Hydropathic analysis revealed hydrophobic regions at the NH2 and COOH termini. The NH2-terminal 13 amino acids probably represent part of the signal peptide, and the COOH-terminal hydrophobic region may act either as a transmembrane anchor or as a signal for attachment to a phosphatidylinositol glycan moiety. The carboxypeptidase M sequence contains six potential Asn-linked glycosylation sites, consistent with its glycoprotein nature. The sequence of carboxypeptidase M was 41% identical with that of the active subunit of human plasma carboxypeptidase N, 41% identical with bovine carboxypeptidase H (carboxypeptidase E, enkephalin convertase), and 15% with either bovine pancreatic carboxypeptidase A or B. Many of the active site residues identified in carboxypeptidases A and B, including all of the zinc-binding residues (2 histidines and a glutamic acid), are conserved in carboxypeptidase M. These data indicate that all of the metallocarboxypeptidases are related, but the nondigestive carboxypeptidases with more specialized functions, present in cell membranes, blood plasma, or secretory granules (i.e., carboxypeptidase M, carboxypeptidase N and carboxypeptidase H), are more closely related to each other (41-49% identity) than they are to carboxypeptidase A or B (15-20% identity).

Human mast cell carboxypeptidase. Purification and characterization.

A carboxypeptidase activity was recently identified in highly purified human lung mast cells and dispersed mast cells from skin. Using affinity chromatography with potato-tuber carboxypeptidase inhibitor as ligand, mast cell carboxypeptidase was purified to homogeneity from whole skin extracts. The purified enzyme yielded a single staining band of approximately 34,500 D on SDS-PAGE. Carboxypeptidase enzyme content estimated by determination of specific activity, was 0.5, 5, and 16 micrograms/10(6) mast cells from neonatal foreskin, adult facial skin, and adult foreskin, respectively. Human mast cell carboxypeptidase resembled bovine carboxypeptidase A with respect to hydrolysis of synthetic dipeptides and angiotensin I, but was distinguished from carboxypeptidase A in its inability to hydrolyze des-Arg9 bradykinin. The amino acid composition of human mast cell carboxypeptidase was similar to the composition of rat mast cell carboxypeptidase. The amino-terminal amino acid sequence of mast cell carboxypeptidase demonstrated 65% positional identity with human pancreatic carboxypeptidase B, but only 19% with human carboxypeptidase A. Thus, human mast cell carboxypeptidase is a novel member of the protein family of zinc-containing carboxypeptidases, in that it is functionally similar but not identical to bovine carboxypeptidase A, but has structural similarity to bovine and human pancreatic carboxypeptidase B.

Isolation and characterization of the cytosolic and chloroplast forms of spinach leaf fructose diphosphate aldolase.

Two different isoenzymes of fructose-P2 aldolase can be resolved by chromatography of crude spinach leaf extracts on DEAE-cellulose columns. The acidic isoenzyme comprises about 85% of the total leaf aldolase activity. The two forms differ in primary structure as judged by their distinctive amino acid compositions, tryptic peptide patterns, and immunological properties. Only the acidic isoenzyme was detected in extracts of isolated chloroplasts, suggesting that this molecule represents the chloroplast form of spinach leaf aldolase while the basic isoenzyme is of cytosolic origin. The cytosolic (basic) isoenzyme and chicken aldolase A4 are similar in the following respects. 1) They have similar specific catalytic activity (10-15 units/mg); 2) they are both highly sensitive to inactivation by very limited digestion with bovine pancreatic carboxypeptidase A; 3) they both have subunit molecular weights of 40,000; 4) they both have derivatized (blocked) NH2-terminal structures; 5) they are both resistant to thermal denaturation at 50 degrees C; and 6) they both regain catalytic activity following reversible denaturation at pH 2.3 or in 5.8 M urea. Also, the cytosolic aldolase cross-reacted immunologically with the single aldolases present in spinach seeds and in wheat germ. Further, this isoenzyme readily "hybridized" with chicken aldolase A4 in vitro. These observations demonstrate the close homology between the cytosolic aldolases derived from plant and animal origins. The chloroplast aldolase had a specific catalytic activity of about 8 units/mg and, like its cytosolic counterpart, was severely inactivated by limited digestion with carboxypeptidase A. However, this isoenzyme was distinct from the cytosolic aldolase in the following characteristics: 1) its "small" subunit size (Mr congruent to 38,000); 2) its underivatized NH2-terminal structure; 3) its high sensitivity to thermal denaturation at 50 degrees C; and 4) its inability to refold into an enzymatically active conformation following denaturation at pH 2.3 or in 5.8 M urea. The distinctive properties of the chloroplast aldolase may be expected for an enzyme which is synthesized as a higher molecular weight precursor on cytosolic polysomes and is then proteolytically processed to the "mature" form during its migration into the chloroplast organelle.

The severed activation segment of porcine pancreatic procarboxypeptidase a is a powerful inhibitor of the active enzyme Isolation and characterisation of the activation peptide

The activation peptide of the monomeric procarboxypeptidase A from porcine pancreas was isolated by means of controlled trypsin digestion of the proenzyme followed by ion-exchange chromatography under dissociating conditions (7 M urea). The molecular weight of the isolated peptide was estimated to be around 11500–12000 (corresponding to approx. 100–103 residues) as judged by SDS electrophoresis and amino acid analysis, a figure that agrees with the difference between the corresponding values for procarboxypeptidase A and carboxypeptidase A (peptidyl- l-amino-acid hydrolase, EC 3.4.17.1). The activation peptide has a high content of hydrophobic and acidic amino acids, and lacks cysteine. A remarkable feature is the strong competitive inhibitory action of the peptide on both porcine and bovine pancreatic carboxypeptidase A activity, with a K i in the nanomolar range, and its null ability to inhibit porcine pancreatic carboxypeptidase B (EC 3.4.17.2). The above properties, and the fact that the peptide has the same N-terminal residue (lysine) as the parent procaboxypeptidase A, suggest that the isolated peptide contains most (if not all) of the activation segment of the proenzyme.

Purification and characterization of carboxypeptidase A from rat skeletal muscle.

Carboxypeptidase A (EC 3.4.17.1) has been purified 44 000-fold in 33% yield from rat skeletal muscle by a four-step procedure. Purification in the presence of dichlorovinyl dimethyl phosphate conveniently inactivates an accompanying chymotrypsin-like enzyme and other serine protease(s) to ensure isolation of pure carboxypeptidase A free of polypeptide contaminants. The enzyme preparation consists of two components with molecular weights of approximately 39 300 and 37 800. The rat muscle carboxypeptidase is very similar to bovine pancreatic carboxypeptidase A in terms of (1) substrate specificity, (2) kinetics and molecular activity, (3) influence of metal ions on catalysis, (4) interaction with inhibitors, (5) effects of ionic strength on activity, and (6) stability and activity as functions of pH. Both muscle and pancreatic carboxypeptidases exhibit enhanced esterolytic activity when assayed in the presence of a variety of indoles and imidazoles or after incubation at relatively high concentrations of MnSO4. The muscle enzyme is substantially less stable than its pancreatic homologue, and in impure preparations is very much less soluble. The latter property is attributable to a binding substance present in such preparations which renders muscle but not pancreatic carboxypeptidase A insoluble until ionic strength is increased to values near 2 M.

Zinc and copper in human nutrition.

of the off-spring of pre<gnant rats fed severely zinc-depleted diets are malformed at term (Hurley, Gowan & Swinerton, 1971). Zinc is also important for normal taste and smell, but the long suggested role of this element in wound healing is less clearly established; at least in man. The presence of copper in almost all forms of living tissue was recognised early in the nineteenth century, but its key role in vertebrate metabolism was not established until 1928 when copper was found to be essential, together with iron, for normal erythropoiesis. Not all the cupro-enzymes so far identified have defined physiological roles, but a number of associations have been recognised in animals and some in man. Defects which affect the cardiovascular system of copper-deficient animals are believed to be related to disorders of cross-linking of the connective tissue proteins caused by deficiency of the cupro-enzyme, lysl oxidase. Disorders of the central nervous system have also been linked to copper deficiency in a number of animal species and lack of the cupro-enzyme tryrosinase is thought to account for the failure of melanin formation, achromotrichia, in animals with pigmented hair, wool, or feathers. Copper is so widely distributed that in humans acquired deficiency has long been regarded as unlikely and it was not until 1972 that studies during total parenteral nutrition (Karpal 8c Peden, 1972) linked copper deficiency in an infant with a syndrome characterised by anaemia, neutropenia and scorbutic type bone lesions. Administration of copper rapidly corrected the haematological abnormalities but the bone defects were slower to respond, although they eventually returned to normal. ZINC Biochemistry In 1940 Keilin & Mann established that zinc was an essential component of the enzyme carbonic anhydrase; but it was not until 1954 that a second enzyme, bovine pancreatic carboxy peptidase A, was also found to contain zinc in an essential biological role. Over the past decade, however, progress has been very

Synthesis of chromophoric, spin label enzyme substrates useful for cryoenzymology.

The spin label nitroxide derivative 3-(2,2,5,5-tetramethylpyrroline-1-oxyl)-propen-2-oic acid has been synthesized and characterized by chemical methods. It is a useful intermediate in the preparation of a new class of chromophoric spin label substrates for enzyme studies, as shown by the synthesis of O-3-(2,2,5,5-tetramethylpyrroline-1-oxyl)-propen-2-oyl-L-beta-phenyllactic acid, a specific ester substrate of bovine pancreatic carboxypeptidase A (peptidyl-L-amino acid hydrolase; EC 3.4.12.2). Kinetic parameters of the esterolytic reaction are conveniently determined by UV spectrophotometric methods, and a reaction intermediate can be stabilized in fluid cryosolvent mixtures at subzero temperatures. Results are presented of preliminary electron spin resonance studies to demonstrate that structural relationships of the spin label substrate in a catalytically active configuration to active site residues can be determined for this low temperature-stabilized reaction intermediate. This substrate thus demonstrates the utility of this new class of spin label derivatives for characterization of enzyme reaction intermediates stabilized by cryoenzymologic techniques.

Structure of 2-hydroxy-5-nitrobenzylated carboxypeptidase A

Bovine pancreatic carboxypeptidase A (EC 3.4.12.2) was treated with dimethyl (2-hydroxy-5-nitrobenzyl)sulfonium chloride at pH 7.5, resulting in a preparation which consisted primarily of a monohydroxynitrobenzylated derivative of the enzyme. Samples of the hydroxynitrobenzylated enzyme were subjected to tryptic digestion and to cyanogen bromide cleavage, and resulting peptides were isolated chromatographically. One tryptic hydroxynitrobenzyl-containing peptide was isolated; its amino acid composition was that of the N-terminal tryptic segment of carboxypeptidase Aγ (residues 8–35). Likewise, CNBr cleavage of the hydroxynitrobenzylated enzyme revealed that the hydroxynitrobenzyl group resided in the N-terminal fragment, F N (residues 8–22). Neither of these hydroxynitrobenzylated peptides contains Trp, the amino acid residue which is characteristically the site of hydroxynitrobenzylation in proteins, and each was found to contain approximately one less Asx than the corresponding native peptide. Both dansylation and automated Edman degradation procedures revealed that the N-terminal Asn of carboxypeptidase Aγ had been modified by hydroxynitrobenzylation of the enzyme. Thus the sulfonium salt reacts with carboxypeptidase A in the same manner as that established earlier for 2-hydroxy-5-nitrobenzyl bromide (Radhakrishnan, T.M., Bradshaw, R.A., Deranleau, D.A. and Neurath, H. (1970) FEBS Lett. 7, 72–76). Such reactivity of the α-amino group presumably reflects its unique location with respect to Trp residues in the tertiary structure of the enzyme.

Catalytic role of the metal ion of carboxypeptidase A in ester hydrolysis.

The mechanism of action of bovine pancreatic carboxypeptidase. Aalpha (peptidyl-L-amino acid hydrolase; EC 3.4.12.2) has been investigated by application of cryoenzymologic methods. Kinetic studies of the hydrolysis of the specific ester substrate O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate have been carried out with both the native and the Co2+-substituted enzyme in the 25 to --45 degrees C temperature range. In the --25 to --45 degrees C temperature range with enzyme in excess, a biphasic reaction is observed for substrate hydrolysis characterized by rate constants for the fast (kf) and the slow (ks) processes. In Arrhenius plots, ks extrapolates to kcat at 25 degrees C for both enzymes in aqueous solution, indicating that the same catalytic rate-limiting step is observed. The slow process is analyzed for both metal enzymes, as previously reported (Makinen, M. W., Yamamura, K., and Kaiser, E. T. (1976) Proc Natl. Acad. Sci. U. S. A. 73, 3882-3886), to involve the deacylation of a mixed anhydride acyl-enzyme intermediate. Near --60 degrees C the acyl-enzyme intermediate of both metal enzymes can be stabilized for spectral characterization. The pH and temperature dependence of ks reveals a catalytic ionizing group with a metal ion-dependent shift in pKa and an enthalpy of ionization of 7.2 kcal/mol for the native enzyme and 6.2 kcal/mol for the Co2+ enzyme. These parameters identify the ionizing catalytic group as the metal-bound water molecule. Extrapolation of the pKa data to 25 degrees C indicates that this ionization coincides with that observed in the acidic limb of the pH profile of log(kcat/Km(app)) for substrate hydrolysis under steady state conditions. The results indicate that in the esterolytic reaction of carboxypeptidase. A deacylation of the mixed anhydride intermediate is catalyzed by a metal-bound hydroxide group.

The pH-dependence of the non-specific esterase activity of carboxypeptidase A

The hydrolysis of the following 6 esters by bovine pancreatic carboxypeptidase A (peptidyl- l-amino-acid hydrolase, EC 3.4.12.2) has been investigated over the range pH 5–10 at 25°C, ionic strength 0.2: CH 3CO 2CHRCO 2H (R = C 6H 5 (ester 3), C 6H 5CH 2 (ester 4)), 4-NO 2C 6H 4CO 2CHRCO 2H (R = C 6H 5 (Ester 5), C 6H 5CH 2 (ester 6), CH 3(CH 2) 2 (ester 7)), CH 3CH 2CO 2CH(CH 2C 6H 5)-CO 2H (ester 9). For each ester the pH dependence of k cat/ K m indicates that substrate binding is controlled by an acid of p K EH = 9.2 ± 0.2 in the free enzyme, and although k cat/ K m decreases in acidic solutions no simple dependence on an enzymic ionization is apparent. For esters 3, 5 and 7 the dependence of k cat on pH is ‘bell-shaped’ and is controlled by p K EH 2S = 6.73, 6.72, 6.23, respectively and p K EHS = 9.3 ± 0.2 for each ester. For esters 4 and 6 the ‘beel-shaped’ k cat (p K EH 2S = 7.38, 6.28, respectively) is modified by a increase in k cat in the vicinity of pH 10. This latter phenomenon is also shown by ester 9, although data are only accessible over the range pH 7–10 for this latter ester due to pronounced product inhibition in acidic solutions. The common pH-dependences observed for the enzymic hydrolyses of these non-specific ester substrates are compared with literature data for specific ester and peptide substrates, and possible assignments for the various enzymic p K a values are discussed.

Studies on the characterization of the Rh o(D) antigen

The Rh o(D) antigen of red cell membranes was solubilized using ethylenediamine tetraacetic acid (EDTA) and 2-mercaptoethanol. The solubilized antigen was partially separated from other solubilized membrane components using molecular filtration. The antigen was treated with various enzymes to learn some of the chemical characteristics. It was found that the activity of the antigen, as measured by hemagglutination inhibition, was not affected by bee venom phospholipase A, Clostridium welchii phospholipase C, calf-intestinal alkaline phosphatase, Vibrio cholerae neuraminidase, pig kidney leucine aminopeptidase, bovine pancreatic carboxypeptidase A, a pig pancreatic carboxypeptidase B. However, the proteolytic enzymes, pronase, trypsin, chymotrypsin and papain, did destroy Rh o(D) activity as measured by hemagglutination inhibition. These results indicate that protein is an important part of the active determinant of the Rh o(D) antigen. The experiments by other investigators have shown that lipid is important to maintain the Rh o(D) activity in the intact membrane; lipid probably helps to maintain the structural conformation of the Rh o(D) molecule in its natural environment. The solubilized Rh o(D) molecules are apparently not dependent on lipid for their Rh o(D) activity.

Further studies of the specificity of carboxypeptidase A towards hippuric acid esters

The kinetics of hydrolysis of a series of 10 new hippurate esters (C6H5CONHCH2CO2CRR1CO2H (I)) by bovine pancreatic carboxypeptidase A have been investigated at pH 7.5, 25 °C, and ionic strength 0.5. Pronounced substrate inhibition was displayed by I: R = H, R1 = C6H5(CH2)2, 3-indolylmethyl, 4-HOC6H4CH2, and 4-FC6H4 whereas pronounced substrate activation was observed for I: R = H, R1 = 4-CH3C6H4, 4-C2H5C6H4, 4-C6H5C6H4, 1-naphthyl, 2-naphthyl, and R = R1 = C2H5. In all cases substrate activation and substrate inhibition were shown to be consistent with ES2 complex formation similar to that previously observed for other hippurate esters. Kinetic parameters were evaluated for each ester and it is noted that ail 13 hippurate esters now known to display substrate inhibition have kcat/Km &gt; 106 M−1 min−1, whereas kcat/km &lt; 106 M−1 min−1 for all 9 hippurate esters known to display substrate activation. The enzymic specificity for the R1 unit of I suggests binding of R1 in a 'bent' hydrophobic pocket having a restricted entrance.

The hydrolysis of esters of N-hippurylglycine and N-pivaloylglycine by carboxypeptidase A

The kinetics of the hydrolysis of five esters of N-hippurylglycine (C 6H 5CONHCH 2CONHCH 2CO 2CRR 1CO 2H ( 2 ) and seven esters of N-pivaloylglycine ((CH 3) 3CCONHCH 2CRR 1CO 2H ( 3 ) by bovine pancreatic carboxypeptidase A (Peptidyl- l-amino-acidhydrolase, EC 3.4.12.2) have been studied at pH 7.5, 25°C and ionic strength 0.5. All N-hippurylglycine esters ( 2 : R  H, R 1  H, C 2H 5, 4-ClC 6H 4, C 6H 5CH 2) display Michaelis-Menten kinetics up to at least 0.1 M substrate. The N-pivaloylglycine esters display either Michaelis-Menten kinetics ( 3 : RH, R 1H, C 2H 5, C 6H 5), substrate activation ( 3 : RH, R 1  4-ClC 6H 4; R  R 1  CH 3) or substrate inhibition ( 3 : R  H, R 1  (CH 3) 2CHCH 2, C 6H 5CH 2). Kinetic parameters have been evaluated for each ester and compared with those for the corresponding hippuric acid esters ( 1 ). The enzymic specificity is shown to be identical for the alcohol moieties of the esters 1 , 2 and 3 and unrelated to the occurrence of substrate activation or inhibition phenomena. These latter phenomena are shown to be characteristic of the enzymic hydrolysis of N-acyl amino acid esters but unimportant for N-acyl dipeptide ester substrates.

Substrate inhibition in the hydrolysis of N-acylglycine esters by carboxypeptidase A

The rates of hydrolysis of a series of 21 N-acylglycine esters (YCONHCH 2CO 2CH(CH 2CH 3)CO 2H ( 2 ) by bovine pancreatic carboxypeptidase A (peptidyl- l-amino-acid hydrolase, EC 3.4.12.2) have been studied over the substrate concentration range 10 −4–10 −1 M at pH 7.5, 25°C, ionic strength 0.5. All substrates display substrate inhibition except Y = CH 3, CH 3CH 2 and (CH 3) 3C for which normal Michaelis-Menten kinetics are observed. In all cases substrate inhibition is consistent with the formation of an ES 2 complex and parameters for the second-degree rate equation v/ E = ( k 2 app S + k 3 app S 2/ K SS app )/( K S app + S + S 2/ K SS app ) have been evaluated. For a series of eight aliphatic groups varying in size between Y = CH 3 and Y = cyclo-C 6H 11 the following linear correlations were observed: -log K S app = 0.82 π + 1.32 and log k 2 app/ K S app = 0.71 π + 5.81 (π is Hansch's hydrophobicity parameter). Aryl and aralkyl Y moieties deviate from these correlation lines. K SS app also depends on the hydrophobicity of Y but no quantitative correlation is obvious. Thus the Y unit of 2 is involved in a hydrophobic interaction with the enzyme when 2 binds at both the catalytically productive and inhibitory sites. Parameters for the enzymic hydrolysis of the esters YCONHCH 2CO 2CH(CH 2CH(CH 3) 2)CO 2H ( 3 ) (Y = C 6H 5(CH 2) n ( n = 0, 1, 2)) are also presented. Pronounced non-productive 1:1 enzyme · substrate complex formation is observed for each of 2 : Y = C 6H 5(CH 2) n ( n = 2,3) and 3 : Y = C 6H 5(CH 2) 2. Hippurate anion is shown to be an uncompetitive inhibitor ( K i = 12 mM) for the hydrolysis of 2 : Y = (CH 3) 3C. Data are now available which can only be interpreted in terms of at least three enzymic sites being available for hydrophobic interactions with ester substrate molecules.