Reversible Inhibition of Carboxypeptidase A. III. Inhibition of Specific Esterase Activity by Substituted Benzoate and Related Anions

1975 ◽  
Vol 53 (13) ◽  
pp. 1984-1992 ◽  
Author(s):  
John W. Bunting ◽  
Chester D. Myers
Keyword(s):  
Ionic Strength ◽  
Esterase Activity ◽  
Competitive Inhibition ◽  
Chemical Properties ◽  
Enzyme Molecule ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Reversible Inhibition ◽  
Noncompetitive Inhibition ◽  
Hydrolysis Of

The reversible inhibition of the hydrolysis of O-hippuryl-L-3-phenyllactic acid by bovine carboxypeptidase A, has been studied for a series of para-substituted benzoate ions (p-XC6H4-CO2−) at pH 7.5, 25°, ionic strength 0.2. For X = H, F, CN, NH2, CH3 competitive inhibition occurs, whereas non-competitive inhibition occurs for X = CF3, NO2, Cl, Br, (CH3)2N, CH3O, (CH3)2CH, (CH3)3C. For X = C2H5 mixed inhibition is observed and this can be separated into individual competitive and noncompetitive components. Uncompetitive inhibition occurs with X = I. The distinction between competitive and noncompetitive inhibition appears to depend on the size of X rather than on its chemical properties. The p-tolylacetate and 3-(p-tolyl)propanoate ions display partially competitive inhibition consistent with the formation of E.I2 species. The inhibition by the 3-(p-iodophenyl)propanoate ion is complex and depends on the binding of at least two inhibitor ions per enzyme molecule.

Reversible Inhibition of Carboxypeptidase A. II. Inhibition of Esterase Activity by Dicarboxylic Acid Anions

1974 ◽  
Vol 52 (11) ◽  
pp. 2053-2063 ◽  
Author(s):  
John W. Bunting ◽  
Chester D. Myers
Keyword(s):  
Dicarboxylic Acid ◽  
Competitive Inhibition ◽  
Dicarboxylic Acids ◽  
Competitive Inhibitor ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Monocarboxylic Acids ◽  
Reversible Inhibition ◽  
Competitive Inhibitors ◽  
Hydrolysis Of

Reversible inhibition of the hydrolysis of O-hippuryl-L-3-phenyllactic acid by carboxypeptidase A has been studied for a series of decarboxylic acids at 25°, pH 7.5, and ionic strength 0.2. All inhibitors studied displayed either strictly competitive or partially competitive inhibition kinetics. For the series CO2H(CH2)nCO2H, strictly competitive inhibition was observed for n = 1, 3, 4, 8, 10, whereas partially competitive inhibition occurs for n = 2, 5, 6, 7. A series of 11 alkyl- and aryl-substituted malonic acids were all strictly competitive inhibitors; for a series of six alkylmalonic acids the inhibition constants are correlated with the Hansch π-parameter by the equation –log K1 = 2.257π + 1.75; arylmalonic acids are poorer inhibitors than expected on the basis of their π-parameters, in accord with a similar observation for monocarboxylic acids. Phthalic acid is a strictly competitive inhibitor (K1 = 1.7 mM), whereas the isomeric isophthalic and terephthalic acids cause relatively little inhibition even at 0.1 M; maleic acid is a partially competitive inhibitor, whereas the isomeric fumaric acid gives only 15% inhibition at 0.1 M. Homophthalic acid and 2,2-dimethyl- and 3,3-dimethylglutaric acids were also investigated.The characteristics of partially competitive inhibition displayed by all dicarboxylic acids and also monomethyl succinate and succinamic acid are consistent with a scheme which assumes the formation of an E.I2 complex. The observed specificity of dicarboxylic acid binding is used to postulate a schematic diagram for binding of these species to the enzyme, and an interpretation of this diagram is suggested on the basis of the crystallographically determined structure of the enzyme.

Reversible Inhibition of the Esterase Activity of Carboxypeptidase A by Carboxylate Anions

1973 ◽  
Vol 51 (16) ◽  
pp. 2639-2649 ◽  
Author(s):  
John W. Bunting ◽  
Chester D. Myers
Keyword(s):  
Competitive Inhibition ◽  
Hydrophobic Interactions ◽  
Enzyme Inhibitor ◽  
Inhibition Kinetics ◽  
Aromatic Rings ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Reversible Inhibition ◽  
Mixed Inhibition ◽  
Carboxylate Anions

Reversible inhibition of the hydrolysis of O-(hippuryl)-L-3-phenyllactic acid by carboxypeptidase A has been studied for 26 carboxylate ion inhibitors at 25°, pH 7.5, and ionic strength 0.2 (NaCl). Competitive inhibition, partially competitive inhibition, and mixed inhibition kinetics were observed. Within homologous series, strictly competitive inhibition by the lower members gave way to partially competitive inhibition with higher members, while homologs having very large hydrocarbon moieties displayed mixed inhibition kinetics. Partially competitive inhibition in this system is not consistent with a scheme involving only 1:1 enzyme–inhibitor complexes; rather, higher order enzyme–inhibitor complexes will be necessary for a complete description of the inhibition mechanism.Inhibition constants (log Ki) for the aliphatic carboxylate ions which are strictly competitive inhibitors, are closely correlated linearly with Hansch's π-parameter for hydrophobicity. This quantitatively confirms the importance of hydrophobic interactions between carboxypeptidase A and inhibiting ions. Carboxylate ions containing aromatic rings are less effective inhibitors than expected on the basis of the π-parameters of their hydrocarbon moieties. The dependence of log Ki on π in this system is unusually strong for a binding phenomenon, and suggests that an inhibitor-dependent conformational change may also be involved.

Effect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B

1975 ◽  
Vol 53 (7) ◽  
pp. 747-757 ◽  
Author(s):  
Graham J. Moore ◽  
N. Leo Benoiton
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Kinetic Behavior ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Carboxypeptidase B ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Basic Peptides ◽  
Hydrolysis Of

The initial rates of hydrolysis of Bz-Gly-Lys and Bz-Gly-Phe by carboxypeptidase B (CPB) are increased in the presence of the modifiers β-phenylpropionic acid, cyclohexanol, Bz-Gly, and Bz-Gly-Gly. The hydrolysis of the tripeptide Bz-Gly-Gly-Phe is also activated by Bz-Gly and Bz-Gly-Gly, but none of these modifiers activate the hydrolysis of Bz-Gly-Gly-Lys, Z-Leu-Ala-Phe, or Bz-Gly-phenyllactic acid by CPB. All modifiers except cyclohexanol display inhibitory modes of binding when present in high concentration.Examination of Lineweaver–Burk plots in the presence of fixed concentrations of Bz-Gly has shown that activation of the hydrolysis of neutral and basic peptides by CPB, as reflected in the values of the extrapolated parameters, Km(app) and keat, occurs by different mechanisms. For Bz-Gly-Gly-Phe, activation occurs because the enzyme–modifier complex has a higher affinity than the free enzyme for the substrate, whereas activation of the hydrolysis of Bz-Gly-Lys derives from an increase in the rate of breakdown of the enzyme–substrate complex to give products.Cyclohexanol differs from Bz-Gly and Bz-Gly-Gly in that it displays no inhibitory mode of binding with any of the substrates examined, activates only the hydrolysis of dipeptides by CPB, and has a greater effect on the hydrolysis of the basic dipeptide than on the neutral dipeptide. Moreover, when Bz-Gly-Lys is the substrate, cyclohexanol activates its hydrolysis by CPB by increasing both the enzyme–substrate binding affinity and the rate of the catalytic step, an effect different from that observed when Bz-Gly is the modifier.The anomalous kinetic behavior of CPB is remarkably similar to that of carboxypeptidase A, and is a good indication that both enzymes have very similar structures in and around their respective active sites. A binding site for activator molecules down the cleft of the active site is proposed for CPB to explain the observed kinetic behavior.

ÉTUDE DES EFFETS ÉLECTROSTATIQUES SUR LE MÉCANISME D'ACTION CATALYTIQUE DE LA PHOSPHATASE ALCALINE INTESTINALE DE VEAU

1965 ◽  
Vol 43 (8) ◽  
pp. 2222-2235 ◽  
Author(s):  
Michel Lazdunski ◽  
Jacques Brouillard ◽  
Ludovic Ouellet
Keyword(s):  
Dielectric Constant ◽  
Ionic Strength ◽  
Maximum Activity ◽  
Enzyme Molecule ◽  
High Substrate Concentration ◽  
Nitrophenyl Phosphate ◽  
Phosphatase Alcaline ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Activated Complex

The influence of dioxane and ethanol on the rate of hydrolysis of p-nitrophenyl phosphate in the presence of an intestinal alcaline phosphatase can be interpreted as a dielectric constant effect, at high substrate concentration. The dielectric constant effect is a function of the pH of the medium and is maximum around pH 9.4 at 25 °C and pH 9.0 at 15 °C. An interpretation suggesting that the change in diameter of the enzyme molecule becoming an activated complex is minimum at a pH of maximum activity is proposed. The same model can take into account the influence of the ionic strength on the same reaction.

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.

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.

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.

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