Substituent Effects in the Carboxypeptidase A Catalyzed Hydrolysis of Substituted L,.beta.-Phenyllactate Esters

The carboxypeptidase A catalyzed hydrolysis of an extensive series of substituted cinnamoyl-L,beta-phenyllactate esters has been investigated. Plots of kcat vs pH are sigmoidal in the pH range 5-9 with an average apparent pKaES of 6.6 +/- 0.1. The values of Km are pH independent in the range pH 5-8. Plots of log kcat/Km vs pH give pKaE values of 6.4 and 9.0 that do not vary significantly through the series. A plot of log kcat (pH 8) vs sigma, the Hammett substituent constant, is linear with a slope rho of 0.5, while log Km vs sigma has a slope of -0.4. The plot of log kcat/Km vs sigma is also linear with rho = 0.9. The Hammett plots are linear at both pH 6 and 8 with closely similar slopes, which indicates that the apparent pKaES near pH 6 does not reflect a change in the rate-determining step. The enzymatic reactions and the nonenzymatic OH- catalyzed hydrolysis reactions are affected alike by changes in the substituent groups; a plot of log kOH, the second-order rate constant for alkaline hydrolysis of the esters, vs log kcat/Km is linear with a slope of 0.9. There is little effect of changing the substituent group in the nonenzymatic pH-independent hydrolysis of the Zn(II) complex of corresponding 4-substituted cinnamic acid 6-carboxypicolinic acid anhydrides (rho < or = 0.1).(ABSTRACT TRUNCATED AT 250 WORDS)