Chang_1980_Biochem.J_187_843

Steady-state kinetics of carboxypeptidase Y, a proteinase from yeast, were studied by using the reaction of 4-nitrophenyl trimethylacetate as a probe. The pH profile of kcat. is sigmoidal in H2O-based buffers for the carboxypeptidase Y-catalysed hydrolysis of this ester (kcat. referring to the rate of deacylation of trimethylacetyl-carboxypeptidase Y). The corresponding pD profile in 2H2O is doubly sigmoidal, with inflexions at pD approximately 3.8 and approximately 6.8. The ionization of pKDapp. approximately 3.8 is caused by a rapid inactivation in 2H2O media by a process that is only slowly reversed on transfer to pH 7.00 phosphate buffer in H2O. The corresponding inactivation in H2O-based buffers of low pH is considerably slower (approximately 30-fold), follows a first-order rate-dependence and is very strongly pH-dependent, indicating some form of co-operative change in enzyme tertiary structure.