Further studies of the specificity of carboxypeptidase A towards hippuric acid esters

Abstract

The kinetics of hydrolysis of a series of 10 new hippurate esters (C6HSCONHCH,C02-CRR,CO,H (I)) by bovine pancreatic carboxypeptidase A have been investigated at p H 7.5, 2S°C, and ionic strength 0.5. Pronounced substrate inhibition was displayed by I : R = H, R , = C6HS(CHZ)2, 3-indolylmethyl, 4-HOC6H,CH,, and 4-FC6H4 whereas pronounced substrate activation was observed for I : R = H , R I = 4-CH3CGH4, 4-C2HSC6H4, 4-C6H5-C6H,, I-naphthyl, 2-naphthyl, and R = R, = C,Hs. In all cases substrate activation and substrate inhibition were shown to be consistent with ES, complex formation similar to that previously observed for other hippurate esters. Kinetic parameters were evaluated for each ester and it is noted that all 13 hippurate esters now known to display substrate inhibition have Ic,,,/Km > lo6 M-' min-I, whereas k,,,/Km < lo6 M-I min-I for all 9 hippurateesters known to display substrate activation. The enzymic specificity for the R , unit of I suggests binding of R, in a 'bent' hydrophobic pocket having a restricted entrance.