Joe Murphy

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

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...

The mechanism of substrate inhibition of the esterase activity of carboxypeptidase A

Abstract Substrate inhibition of the hydrolysis of esters of hippuric acid by carboxypeptidase A is consistent with the formation of an E.S2 complex which, independent of the alcohol moiety of the ester, reacts approximately 26 times more slowly than E.S complex. The binding of a second substrate molecule to E.S also does not depend significantly on the structure of the alcohol portion of the ester.