Chang W T, Douglas K T
Biochem J. 1980 Jun 1;187(3):843-9. doi: 10.1042/bj1870843.
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.
利用4-硝基苯基三甲基乙酸酯的反应作为探针，研究了酵母蛋白酶羧肽酶Y的稳态动力学。在基于水的缓冲液中，羧肽酶Y催化该酯水解时，kcat的pH曲线呈S形（kcat指三甲基乙酰羧肽酶Y的脱酰化速率）。在2H2O中相应的pD曲线呈双S形，在pD约为3.8和约6.8处有拐点。pKDapp约为3.8的电离是由2H2O介质中的快速失活引起的，该过程在转移至pH 7.00的H2O磷酸盐缓冲液中时仅缓慢逆转。在低pH的基于水的缓冲液中的相应失活要慢得多（约30倍），遵循一级速率依赖性，并且对pH非常敏感，表明酶三级结构存在某种形式的协同变化。
