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 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 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.

Kinetic studies of modifier effects on the carboxypeptidase A catalyzed hydrolyses of peptides

1987 ◽  
Vol 65 (8) ◽  
pp. 717-725 ◽  
Author(s):  
John F. Sebastian ◽  
Richard S. Hinks ◽  
Ralf V. Reuland
Keyword(s):  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Peptidase Activity ◽  
Benzene Sulfonate ◽  
Carboxypeptidase A ◽  
Structural Requirements ◽  
Detailed Mechanism ◽  
Hydrolysis Of ◽  
Phenyl Alanine ◽  
Standing Question

A variety of modifiers of carboxypeptidase A (CPA) have been investigated in an effort to understand the structural requirements of inhibitors and activators of peptidase activity. It is proposed that an understanding of the mechanism of action of reversible activators of the enzyme may bear on the long standing question of whether the detailed mechanism of peptidase activity is different from that of esterase activity. An analog of the activator 2,2-dimethyl-2-silapentane-5-sulfonate, 5,5-dimethylhexanoate, was found to be a competitive inhibitor of the CPA-catalyzed hydrolysis of benzoylglycyl-L-phenyl-alanine. The modifier 4-phenyl-3-butenoate (styrylacetic acid) was determined to be an activator. The sulfonates benzene-sulfonate, p-toluenesulfonate, phenylmethanesulfonate, 2-phenylethanesulfonate, and 3-phenylpropanesulfonate were all found to be activators.

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.

Kinetics of the Inhibition of Plasmin in Acidified Human Plasma

1982 ◽  
Vol 48 (03) ◽  
pp. 257-259 ◽  
Author(s):  
H R Lijnen ◽  
M Maes ◽  
M Castel ◽  
M Samama ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Plasma Proteins ◽  
Competitive Inhibitor ◽  
Normal Plasma ◽  
Competitive Inhibitors ◽  
Rate Of Hydrolysis ◽  
Kinetics Of ◽  
Hydrolysis Of

SummaryAcid-treated human plasma is a competitive inhibitor of the hydrolysis of D-Val-Leu-Lys-Nan (S-2251) by plasmin. The rate of hydrolysis is decreased to 50% by 750 fold diluted acidified normal plasma and by 60 fold diluted acidified α2-antiplasmin depleted plasma (α2-antiplasmin concentration less than 2%). These findings suggest that α2-antiplasmin is a contributary but not the main competitive inhibitor of acidified plasma. This interpretation is supported by the finding that α2-antiplasmin depleted plasma reconstituted with purified α2-antiplasmin inhibits the hydrolysis of S-2251 by plasmin at a 125 fold dilution following acidification and by the finding that in a purified system acid inactivated α2-antiplasmin inhibits the hydrolysis of S-2251 by plasmin with a Ki of 25 nM. Thus, besides α2-antiplasmin, other plasma proteins which are at least in part eliminated by the removal of α2-antiplasmin from plasma by immunoadsorption appear to be competitive inhibitors for plasmin in acidified plasma. It is suggested that several competitive inhibitors for plasmin are present and/or generated in acidified plasma and that these inhibitors may at least in part be responsible for the variability in the results of measurements of plasminogen and/or plasmin in plasma following acidification.

Diagnostic Enzymology of a-L-Iduronidase with Special Reference to a Sulphated Disaccharide Derived from Heparin

1982 ◽  
Vol 62 (2) ◽  
pp. 193-201 ◽  
Author(s):  
J. J. Hopwood ◽  
Vivienne Muller
Keyword(s):  
Enzyme Activity ◽  
Competitive Inhibitor ◽  
Ph Profile ◽  
Ph Values ◽  
Substrate Structure ◽  
Cultured Skin ◽  
Bivalent Cations ◽  
Competitive Inhibitors ◽  
Specific Ion Effect ◽  
Hydrolysis Of

1. Iduronosyl anhydro[1-3H]mannitol 6-sulphate (IMs), iduronosyl anhydro[1-3H]mannitol, phenyl iduronide (PhI) and 4-methylumbelliferyl iduronide have been compared as substrates for the diagnostic estimation of α-l-iduronidase activity present in human leucocyte and cultured skin fibroblast homogenates. The pH profile of leucocyte and fibroblast iduronidase activity was dependent on substrate structure and concentration, the ionic strength and the nature of the buffer ion used in the assay mixture. 2. NaCl, KBr and Na2SO4 were shown to be parabolic competitive inhibitors of IMs activity, the K1 with fibroblast homogenates being 34, 13.4 and 0.22 mmol/l respectively. NaCl and KBr were shown to have a primary salt effect on the interaction between enzyme and substrate but Na2SO4 appeared to have a specific ion effect at a cationic binding site. 3. NaCl inhibited the hydrolysis of IMs at all pH values studied, whereas NaCl concentrations of 0.2 mol/l inhibited the hydrolysis of PhI at pH values below 3.8 but activated the enzyme at higher incubation pH values. 4. Cu2+ was shown to be a potent non-competitive inhibitor of IMs enzyme activity with an apparent Kl, of approximately 0.02 mmol/l. The enzyme activity was inhibited by Fe2+ (Kl 4 mmol/l), Hg2+ and Ag+, but has not significantly been affected by other univalent or bivalent cations. 5. The presence of solvent and salt effects on apparent Km but not the Vmax. suggest that the binding of IMs to the enzyme involved charge neutralization, and it is inferred that two cationic binding sites are present at the active site. It is postulated that one site specifically binds to the iduronic acid carboxyl group, the other to the 6-sulphate of the anhydromannitol moiety.

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.

Sign in / Sign up

Export Citation Format

Share Document