Substrate Activation in the Carboxypeptidase A Catalysis of Ester Hydrolysis

1974 ◽  
Vol 52 (23) ◽  
pp. 3829-3836 ◽  
Author(s):  
Joe Murphy ◽  
John W. Bunting
Keyword(s):  
Phenyl Ring ◽  
Mandelic Acid ◽  
Substrate Inhibition ◽  
Carboxypeptidase A ◽  
Substrate Activation ◽  
Hydrolysis Of ◽  
Derivatives Of ◽  
Substrate Concentrations ◽  
Acid Unit ◽  
Reaction Schemes

The hydrolyses of the O-hippuryl derivatives of glycolic acid (1a), 2-methyllactic acid (1b), and p-chloromandelic acid (1c) by bovine carboxypeptidase A display substrate activation. The hydrolyses of the latter two esters also display substrate inhibition at high substrate concentrations (>0.03 and >0.05 M respectively). Partial kinetic analyses are presented, and these phenomena are discussed in terms of reaction schemes which involve substrate binding at both activating and inhibiting regulatory sites.The hydrolysis of 1b by this enzyme is the first indication that the presence of a hydrogen atom on the α-carbon atom of the alcohol moiety is not obligatory for ester substrates of carboxypeptidase A. The binding of 1c at the catalytic site is approximately 1000 times weaker than for O-hippurylmandelic acid and indicates a dramatic influence for the p-chloro substituent on the binding of the phenyl ring of the mandelic acid unit.

scholarly journals Substrate Inhibition in the Hydrolysis of Hippuric Acid Esters by Carboxypeptidase A

1975 ◽  
Vol 53 (2) ◽  
pp. 283-294 ◽  
Author(s):  
Joe Murphy ◽  
John W. Bunting
Keyword(s):  
Substrate Concentration ◽  
Hippuric Acid ◽  
Substrate Inhibition ◽  
Competitive Inhibitor ◽  
Side Chain ◽  
Carboxypeptidase A ◽  
Substrate Activation ◽  
Hydrolysis Of ◽  
Substrate Concentrations ◽  
Acid Esters

The dependence of initial velocity upon substrate concentration has been examined in the carboxypeptidase A catalyzed hydrolysis of the following hippuric acid esters (at pH 7.5, 25°, ionic strength O.5): C6H5CONHCH2CO2CHRCO2H: R=CH3; CH2CH3;(CH2)2CH3; (CH2)3CH3; (CH2)5CH3; CH(CH3)2; CH2CH(CH3)2; C6H5; CH2C6H5. All of these esters display marked substrate inhibition of their enzymic hydrolyses. With the exception of R=CH3, the velocity-substrate concentration profiles for each of these esters can be rationalized by the formation of an E.S2 complex which, independent of the alcohol moiety of the ester, reacts approximately 25 times more slowly than the E.S complex. For most of these esters, the formation of E.S2 approximates ordered binding of the substrate molecules at the catalytic and inhibitory sites. While binding at the catalytic site is markedly dependent on the nature of the R group, binding of a second substrate molecule to E.S is not significantly affected by the nature of the R side chain. For R=C6H5, the D ester is neither a substrate nor a competitive inhibitor of the hydrolysis of the L-ester but can replace the L-ester at the binding site which is responsible for substrate inhibition. The kinetic analysis suggests that this behavior of D and L -enantiomers is also typical of the other esters examined (except possibly R=CH3). For R=CH3 only, substrate activation also seems to occur prior to the onset of substrate inhibition at higher substrate concentrations.

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

1978 ◽  
Vol 56 (16) ◽  
pp. 2188-2193
Author(s):  
John W. Bunting ◽  
Samuel S.-T. Chu
Keyword(s):  
Ionic Strength ◽  
Hippuric Acid ◽  
Substrate Inhibition ◽  
Carboxypeptidase A ◽  
Hydrophobic Pocket ◽  
Substrate Activation ◽  
Kinetics Of Hydrolysis ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
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 > 106 M−1 min−1, whereas kcat/km < 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 Related to O-Hippuryl-2-hydroxybutanoic Acid by Carboxypeptidase A

1974 ◽  
Vol 52 (14) ◽  
pp. 2640-2647 ◽  
Author(s):  
John W. Bunting ◽  
Joe Murphy
Keyword(s):  
Ionic Strength ◽  
Substrate Inhibition ◽  
Binding Modes ◽  
Carboxypeptidase A ◽  
Acid Moiety ◽  
Hydroxybutanoic Acid ◽  
Hydrolysis Of Esters ◽  
Hydrolysis Of ◽  
Acid Esters

The hydrolysis of each of the following esters by bovine carboxypeptidase A has been studied at pH 7.5, 25°, ionic strength 0.5: O-hippuryl-, O-phenaceturyl-, O-aceturyl-, O-(N-methylhippuryl)-, and O-(N-hippurylglycyl)-2-hydroxybutanoic acids, and 2-(3-benzoylpropanoxy)-, 2-benzoxyacetoxy-, and 2-(4-phenylbutanoxy)butanoic acids. Substrate inhibition occurs with only the hippuric and phenaceturic acid esters and in the six other cases simple Michaelis–Menten kinetics are observed. The relatively minor variations in the structures of the acid moieties of these esters lead to quite large variations in Km, although kcat seems to be relatively independent of the nature of the acid moiety. Binding modes of substrate molecules at both the catalytic and inhibitory sites are discussed in the light of these observations.

Inhibition of carboxypeptidase A catalyzed peptide hydrolysis by 3-phenylpropanoate at activating and nonactivating substrate concentrations

1979 ◽  
Vol 57 (4) ◽  
pp. 357-365
Author(s):  
Roger Poorman ◽  
Maria Kuo ◽  
Douglas I. Johnson ◽  
Sharon Lin ◽  
John F. Sebastian
Keyword(s):  
Kinetic Mechanism ◽  
Carboxypeptidase A ◽  
Peptide Hydrolysis ◽  
Substrate Activation ◽  
Noncompetitive Inhibitor ◽  
A Site ◽  
Substrate Concentrations

The carboxypeptidase A catalyzed hydrolyses of five structurally related dipeptide substrates in the presence of the inhibitor 3-phenylpropanoate have been studied. At nonactivating substrate concentrations, 3-phenylpropanoate is a mixed inhibitor of carbobenzoxyglycyl-L-phenylalanine hydrolysis and a noncompetitive inhibitor of the hydrolyses of benzoylglycyl-L-phenylalanine, cinnamoylglycyl-L-phenylalanine, hydrocinnamoylglycyl-L-phenylalanine, and acetylglycyl-L-phenylalanine. When carbobenzoxyglycyl-L-phenylalanine and benzoylglycyl-L-phenylalanine exhibit substrate activation, inhibition by 3-phenylpropanoate is mixed but appears to be mostly competitive. Proposed here is a site for the binding of 3-phenylpropanoate along with a kinetic mechanism consistent with these data.

Reversible Inhibition of Carboxypeptidase A. IV. Inhibition of Specific Esterase Activity by Hippuric Acid and Related Species and other Amino Acid Derivatives and a Comparison with Substrate Inhibition

1975 ◽  
Vol 53 (13) ◽  
pp. 1993-2004 ◽  
Author(s):  
John W. Bunting ◽  
Chester D. Myers
Keyword(s):  
Amino Acids ◽  
Hippuric Acid ◽  
Substrate Inhibition ◽  
Enzymic Hydrolysis ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Peptide Substrates ◽  
Uncompetitive Inhibition ◽  
Hydrolysis Of ◽  
Noncompetitive Inhibitors

The anions of each of the following carboxylic acids exhibit uncompetitive inhibition of the hydrolysis of O-hippuryl-L-3-phenyllactic acid by bovine carboxypeptidase A at pH 7.5, 25°, ionic strength 0.2: hippuric acid, p-chloro- and p-nitrohippuric acids, hippurylglycine, carbobenzoxyglycine, phenaceturic acid, N'-(3-phenylpropanoyl)glycine, benzoxyacetic acid, 3-benzoylpropanoic acid, and O-hippuryl-D-mandelic acid. In each case, this uncompetitive inhibition is consistent with the ordered binding of substrate and inhibitor to the enzyme; i.e. the inhibitor binds to E.S but not to the free enzyme. Evidence is presented for the binding site for uncompetitive inhibitors being the same as for inhibitory ester substrate molecules. Comparison of the specificities of uncompetitive inhibitors and esters which display substrate inhibition provides evidence for a critical conformational change which controls the binding of uncompetitive inhibitors and inhibitory substrate molecules.D-Phenylalanine, D-leucine, D-p-nitrophenylalanine, glycyl-L-tyrosine, glycyl-L-phenylalanine, and glycyl-L-leucine are competitive inhibitors of the enzymic hydrolysis of O-hippuryl-L-3-phenyllactic acid, whereas the N-chloroacetyl derivatives of L-tyrosine, L-phenylalanine, and L-leucine are noncompetitive inhibitors. For the above D-amino acids, glycyl dipeptides, and N-chloroacetyl amino acids, the phenylalanine derivative in each case is a considerably stronger inhibitor than the corresponding leucine derivative. This preference is similar to that observed for the binding of peptide substrates but the reverse of that observed for ester substrates and simple mono- and dicarboxylate ion inhibitors.The peptide substrates carbobenzoxyglycylglycyl-L-phenylalanine and N-chloroacetyl-L-phenylalanine are noncompetitive inhibitors of the enzymic hydrolysis of O-hippuryl-L-3-phenyllactic acid. This clearly demonstrates the presence of different ester and peptide binding sites in this enzyme, which is consistent with conclusions from recent studies in other laboratories.

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