SUBSTITUENT EFFECTS IN THE CARBOXYPEPTIDASE-A CATALYZED-HYDROLYSIS OFSUBSTITUTED L,BETA-PHENYLLACTATE ESTERS

The carboxypeptidase A catalyzed hydrolysis of an extensive series of substituted cinnamoyl-L,beta-phenyllactate esters has been investigate d. Plots of k(cat) vs pH are sigmoidal in the pH range 5-9 with an ave rage apparent pK(a)(ES) of 6.6 +/- 0.1. The values of K-m are pH indep endent in the range pH 5-8. Plots of log k(cat)/K-m vs pH give pK(a)(E ) values of 6.4 and 9.0 that do not vary significantly through the ser ies. A plot of log k(cat) (pH 8) vs sigma, the Hammett substituent con stant, is linear with a slope rho of 0.5, while log K-m vs sigma has a slope of -0.4. The plot of log k(cat)/K-m vs sigma is also linear wit h rho = 0.9. The Hammett plots are linear at both pH 6 and 8 with clos ely similar slopes, which indicates that the apparent pK(a)(ES) near p H 6 does not reflect a change in the rate-determining step. The enzyma tic reactions and the nonenzymatic OH- catalyzed hydrolysis reactions are affected alike by changes in the substituent groups; a plot of log k(OH), the second-order rate constant for alkaline hydrolysis of the esters, vs log k(cat)/K-m is linear with a slope of 0.9. There is litt le effect of changing the substituent group in the nonenzymatic pH-ind ependent hydrolysis of the Zn(II) complex of corresponding 4-substitut ed cinnamic acid 6-carboxypicolinic acid anhydrides (rho less than or equal to 0.1). The carboxypeptidase A catalyzed hydrolysis of (alpha-b enzoylamino)cinnamoyl-L,beta-phenyllactate at pH values from 7.5 to 9. 5 produces no absorbance changes in the range 340-370 nm that would be characteristic of the oxazolinone formed by attack of the neighboring benzoylamino group on an anhydride intermediate. Also, there is only a small effect of high concentrations of fluoride ion on the reactions of that ester. The above results do not support a mechanism involving rate-determining hydrolysis of an anhydride intermediate in the react ions of the benzoylamino derivative. If an anhydride occurs as an inte rmediate in the reactions of the 4-substituted cinnamoyl esters, then it does not build up. A mechanism involving the attack of Zn(II)-bound OH- on the ester substrates is considered as an alternative to nucleo philic attack by Glu-270.