The pH-dependence of the non-specific esterase activity of carboxypeptidase A

Abstract

The hydrolysis of the following 6 esters by bovine pancreatic carboxypeptidase A (peptidyl- l-amino-acid hydrolase, EC 3.4.12.2) has been investigated over the range pH 5–10 at 25°C, ionic strength 0.2: CH 3CO 2CHRCO 2H (R = C 6H 5 (ester 3), C 6H 5CH 2 (ester 4)), 4-NO 2C 6H 4CO 2CHRCO 2H (R = C 6H 5 (Ester 5), C 6H 5CH 2 (ester 6), CH 3(CH 2) 2 (ester 7)), CH 3CH 2CO 2CH(CH 2C 6H 5)-CO 2H (ester 9). For each ester the pH dependence of k cat/ K m indicates that substrate binding is controlled by an acid of p K EH = 9.2 ± 0.2 in the free enzyme, and although k cat/ K m decreases in acidic solutions no simple dependence on an enzymic ionization is apparent. For esters 3, 5 and 7 the dependence of k cat on pH is ‘bell-shaped’ and is controlled by p K EH 2S = 6.73, 6.72, 6.23, respectively and p K EHS = 9.3 ± 0.2 for each ester. For esters 4 and 6 the ‘beel-shaped’ k cat (p K EH 2S = 7.38, 6.28, respectively) is modified by a increase in k cat in the vicinity of pH 10. This latter phenomenon is also shown by ester 9, although data are only accessible over the range pH 7–10 for this latter ester due to pronounced product inhibition in acidic solutions. The common pH-dependences observed for the enzymic hydrolyses of these non-specific ester substrates are compared with literature data for specific ester and peptide substrates, and possible assignments for the various enzymic p K a values are discussed.