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.

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.

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 Two invertebrate acetylcholinesterases show activation followed by inhibition with substrate concentration

1998 ◽  
Vol 329 (2) ◽  
pp. 329-334 ◽  
Author(s):  
Véronique MARCEL ◽  
Laurent Gagnoux PALACIOS ◽  
Christophe PERTUY ◽  
Patrick MASSON ◽  
Didier FOURNIER
Keyword(s):  
Drosophila Melanogaster ◽  
Caenorhabditis Elegans ◽  
Substrate Concentration ◽  
Substrate Activation ◽  
Excess Substrate ◽  
Catalytic Behaviour ◽  
Increased Activity ◽  
Substrate Concentrations ◽  
Single Enzyme

In vertebrates there are two cholinesterases, with differences in catalytic behaviour with respect to substrate concentration: butyrylcholinesterase displays an increased activity at low substrate concentrations, whereas acetylcholinesterase displays inhibition by excess substrate. In two invertebrates, Drosophila melanogaster and Caenorhabditis elegans, we found cholinesterases that showed both kinetic complexities: substrate activation at low substrate concentrations followed by inhibition at higher concentrations. These triphasic kinetics can be explained by the presence of two enzymes with different kinetic behaviours or more probably by the existence of a single enzyme regulated by the substrate concentration.

scholarly journals Structure and Dynamics of the Metal Site of Cadmium-Substituted Carboxypeptidase A in Solution and Crystalline States and under Steady-State Peptide Hydrolysis†

Biochemistry ◽  
1997 ◽  
Vol 36 (38) ◽  
pp. 11514-11524 ◽  
Author(s):  
Rogert Bauer ◽  
Eva Danielsen ◽  
Lars Hemmingsen ◽  
Marianne V. Sørensen ◽  
Jens Ulstrup ◽  
...  
Keyword(s):  
Steady State ◽  
Carboxypeptidase A ◽  
Peptide Hydrolysis ◽  
Metal Site ◽  
Structure And Dynamics

scholarly journals Stimulation of microsomal uridine diphosphate glucuronyltransferase by glucuronic acid derivatives

1974 ◽  
Vol 139 (1) ◽  
pp. 243-249 ◽  
Author(s):  
Donald A. Vessey ◽  
David Zakim
Keyword(s):  
Metal Ions ◽  
Glucuronic Acid ◽  
Kinetic Mechanism ◽  
Prior Treatment ◽  
Acid Activation ◽  
Uridine Diphosphate ◽  
High Concentrations ◽  
A Site ◽  
Stimulation Of

The glucuronic acid adducts of 1-naphthol, 2-naphthol and 4-methylumbelliferone activate microsomal UDP-glucuronyltransferase (EC 2.4.1.17) when the enzyme is assayed with p-nitrophenol as aglycone. Phenyl glucuronide and oestriol 3β-glucuronide also activate UDP-glucuronyltransferase. but to a lesser extent. Activation by glucuronides is not dependent on metal ions, but is blocked by prior treatment of microsomal fractions with p-chloromercuribenzoate. The kinetic mechanism of activation is concluded to be an increase in the affinity of the enzyme for UDP-glucuronic acid. Activation by 1-naphthyl glucuronide, at high concentrations of p-nitrophenol, is not affected by 1-naphthol. Apparently 1-naphthyl glucuronide activates the preparation by binding at a site that is separate from the site of glucuronidation of 1-naphthol. Further evidence for the existence of distinct effector sites for the glucuronides was provided by the finding that activation by glucuronides is inhibited competitively by aglycone glucosides. These glucosides do not inhibit the rate of glucuronidation of p-nitrophenol in the absence of glucuronide adducts, nor do they alter the rate of glucuronidation of 1-naphthol. When UDP-glucuronyltransferase is assayed with 1-naphthol as aglycone it is activated by p-nitrophenyl glucuronide, 4-methyl-umbelliferyl glucuronide and under appropriate conditions by its own glucuronide. These activations are similarly inhibited by aglycone glucosides. p-Nitrophenyl glucuronide also stimulates the rate of glucuronidation of o-aminophenol, o-aminobenzoate and bilirubin.

Parallelism between ethanol and imidazole interactions with horse liver alcohol dehydrogenase

1978 ◽  
Vol 56 (11) ◽  
pp. 1016-1020 ◽  
Author(s):  
Patricia A. Collins ◽  
Charles S. Hanes ◽  
J. Tze-Fei Wong
Keyword(s):  
Alcohol Dehydrogenase ◽  
Binding Site ◽  
Kinetic Mechanism ◽  
Free Enzyme ◽  
Horse Liver Alcohol Dehydrogenase ◽  
Liver Alcohol Dehydrogenase ◽  
Rate Effects ◽  
Horse Liver ◽  
Substrate Concentrations ◽  
Enzyme Species

The rate effects of imidazole on the EE isoenzyme of horse liver alcohol dehydrogenase have been analysed in terms of the elucidated kinetic mechanism of the enzyme. These imidazole effects on both directions of the reaction within nonexcess as well as excess ranges of substrate concentrations pointed to the competition between imidazole and ethanol for binding to the same three enzyme species in the kinetic mechanism, namely the free enzyme, the enzyme–NAD+ complex, and the enzyme–NADH complex. Moreover, both imidazole and ethanol brought about an enhancement in the rate of dissociation of NAD+ from its binding site on the enzyme.

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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