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

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 degrees C, ionic strength 0.2: CH3CO2CHRCO2H (R = C6H5 (ester 3), C6H5CH2 (ester 4), 4-NO2C6H4CO2CHRCO2H (R = C6H5 (ester 5), C6H5CH2 (ester 6), CH3(CH2)2 (ester 7)), CH3CH2CO2CH(CH2C6H5)-CO2H (ester 9). For each ester the pH dependence of kcat/Km indicates that substrate binding is controlled by an acid of pKEH = 9.2 +/- 0.2 in the free enzyme, and although kcat/Km decreases in acidic solutions no simple dependence on an enzymic ionization is apparent. For esters 3, 5 and 7 the dependence of kcat on pH is 'bell-shaped' and is controlled by pKEH2S = 6.73, 6.72, 6.23, respectively and pKEHS = 9.3 +/- 0.2 for each ester. For esters 4 and 6 the 'bell-shaped' kcat (pKEH2S = 7.38, 6.28, respectively) is modified by a significant increase in kcat 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 nonspecific ester substrates are compared with literature data for specific ester and peptide substrates, and possible assignments for the various enzymic pKa values are discussed.