Phillips M A, Hedstrom L, Rutter W J
Hormone Research Institute, University of California, San Francisco 94143-0534.
Protein Sci. 1992 Apr;1(4):517-21. doi: 10.1002/pro.5560010406.
Arg-127 stabilizes the oxyanion of the tetrahedral intermediate formed during Zn2+ carboxypeptidase A-catalyzed hydrolysis. Mutant carboxypeptidases lacking Arg-127 exhibit substantially reduced rates of hydrolysis with the change manifest almost entirely in kcat (kcat/Km is decreased by 10(4) for R127A). Therefore, Arg-127 stabilizes the enzyme-transition state complex but not the ground state enzyme-substrate complex (Phillips, M.A., Fletterick, R., & Rutter, W.J., 1990, J. Biol. Chem. 265, 20692-20698). The addition of guandine, methylguanidine, or ethylguanidine to R127A increases the kcat for hydrolysis of Bz-gly(o)phe by 10(2) without changing the Km. Dissociation constants (Kd) for the guanidine derivatives range from 0.1 to 0.5 M. The binding affinity for the transition state analog Cbz-phe-alaP(o)ala is increased similarly by 10(2); in contrast, the binding affinity of the ground state inhibitor benzylsuccinic acid is not altered. Thus, guanidine derivatives mimic Arg-127 in stabilizing the rate-limiting transition state. Hydrolysis of Bz-gly-(o)phe by wild-type carboxypeptidase, R127K, or R127M is not substantially affected by guanidine derivatives. Additionally, primary amines do not change the activity of R127A. These observations imply that guanidine binds in the cavity vacated by Arg-127 specifically and in a productive conformation for catalysis.
精氨酸-127可稳定锌离子羧肽酶A催化水解过程中形成的四面体中间体的氧阴离子。缺乏精氨酸-127的突变羧肽酶水解速率大幅降低，这种变化几乎完全体现在催化常数（kcat）上（对于R127A，kcat/Km降低了10^4）。因此，精氨酸-127稳定酶-过渡态复合物，而非基态酶-底物复合物（菲利普斯，M.A.，弗莱泰里克，R.，& 拉特，W.J.，1990，《生物化学杂志》265，20692 - 20698）。向R127A中添加胍、甲基胍或乙基胍可使Bz - gly(o)phe水解的kcat增加10^2，而不改变Km。胍衍生物的解离常数（Kd）范围为0.1至0.5 M。对过渡态类似物Cbz - phe - alaP(o)ala的结合亲和力同样增加10^2；相比之下，基态抑制剂苄基琥珀酸的结合亲和力未改变。因此，胍衍生物在稳定限速过渡态方面模拟了精氨酸-127。野生型羧肽酶、R127K或R127M对Bz - gly-(o)phe的水解不受胍衍生物的显著影响。此外，伯胺不会改变R127A的活性。这些观察结果表明，胍以特定的、有利于催化的构象结合在精氨酸-127腾出的空腔中。
