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)
Osumi
A
A
Department of Biochemistry, University of Southern California, Los Angeles 90033.
Rahmo
A
A
King
S W
SW
Przystas
T J
TJ
Fife
T H
TH
D002268:Carboxypeptidases / Q000378:metabolism*; D043422:Carboxypeptidases A; D002384:Catalysis; D004952:Esters / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D007773:Lactates / Q000378:metabolism*; D008956:Models, Chemical; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D004952:Esters; D007773:Lactates; C039029:O-cinnamoyl beta-phenyllactate; D002268:Carboxypeptidases; D043422:Carboxypeptidases A