The pH dependence of the hydrolysis of hippuric acid esters by carboxypeptidase A

Abstract

The pH dependence (pH 4.5-10.5) of the hydrolysis of 7 hippuric acid esters (C6H5CONHCH2C-O2CR1R2CO2H: 1a: R1 = R2 = H; 1b: R1 = R2 = CH3; 1c: R1 = H, R2 = p-ClC6H4; 1d: R1 = H, R2 = C2H5; 1e: R1 = H, R2 = (CH3)2CHCH2; 1f: R1 = H, R2 = C6H5; 1g: R1 = H, R2 = C6H5CH2) by bovine carboxypeptidase A was investigated, and the pH dependence of the substrate activation of 1a-c and the substrate inhibition of 1d-g were compared. For all 7 esters the catalytically productive binding of the first substrate molecule depends on enzymatic pKa values of 6.0 and 9.1. For 1d, 1e, and 1g the hydrolysis rate (k2app) of this complex is pH independent, whereas for 1f, k2app depends on a pKa of 5.9. The hydrolysis rate (k3app) of the 1:2 enzyme-substrate complex (ES2) is pH independent for 1d-g, but for 1a-c, k3app depends on the following pKa values: 1a, 6.1 and 9.1; 1b, 5.4; 1c, 6.6. The pH dependences of k2app for 1f and k3app for 1c are rationalized by the presence of catalytically nonproductive species. Equivalent ES2 species are believed to be productive for 1c-g; the productive ES2 species for 1b must be quiet different.