Cornish-Bowden A J, Greenwell P, Knowles J R
Biochem J. 1969 Jun;113(2):369-75. doi: 10.1042/bj1130369.
To delineate further the pathway of pepsin-catalysed reactions, three types of experiments were performed: (a) the enzyme-catalysed hydrolysis of a number of di- and tri-peptide substrates was studied with a view to observing the rate-determining breakdown of a common intermediate; (b) the interaction of pepsin with several possible substrates for which ;burst' kinetics might be expected was investigated; (c) attempts were made to trap a possible acyl-enzyme intermediate with [(14)C]methanol in both a hydrolytic reaction (with N-acetyl-l-phenylalanyl-l-phenylalanylglycine) and in a ;virtual' reaction (with N-acetyl-l-phenylalanine) under conditions where extensive hydrolysis or (18)O exchange is known to occur. It is concluded that (i) intermediates in pepsin-catalysed reactions (aside from the Michaelis complex) occur subsequently to the rate-determining transition state, and (ii) an acyl-enzyme intermediate, if such is formed, cannot be trapped with [(14)C]methanol in these systems.
为了进一步阐明胃蛋白酶催化反应的途径，进行了三种类型的实验：(a) 研究了胃蛋白酶催化多种二肽和三肽底物的水解反应，以观察常见中间体的限速分解；(b) 研究了胃蛋白酶与几种可能预期出现“爆发”动力学的底物的相互作用；(c) 试图在水解反应（用N-乙酰-L-苯丙氨酰-L-苯丙氨酰甘氨酸）和“虚拟”反应（用N-乙酰-L-苯丙氨酸）中，在已知会发生广泛水解或(18)O交换的条件下，用[(14)C]甲醇捕获可能的酰基酶中间体。得出的结论是：(i) 胃蛋白酶催化反应中的中间体（除米氏复合物外）在限速过渡态之后出现，并且 (ii) 如果形成酰基酶中间体，在这些系统中不能用[(14)C]甲醇捕获。
