Vajda T, Náray-Szabó G
Institute of Organic Chemistry, Eötvös University, Budapest, Hungary.
Acta Biochim Biophys Hung. 1988;23(2):195-202.
The Asp102-carboxylate negative charge of the trypsin catalytic-triad has been substituted in part by Cl- counter-ions. A His57-imidazolium cation and Ser195-tetrahedral oxyanion ionpair generated in the acylation step of catalysis is stabilized by the negative charge of Asp102. The importance of correct location of this negative charge has been investigated by experimental analysis of the NaCl influence on the acylation-rate of trypsin, as well as by electrostatic, potential calculations. The acylation-rate was determined with stopped-flow technique under pseudo-first order conditions, by using 4-nitrophenyl-4'-guanidinium benzoate active site titrant at pH 4.25 +/- 0.04, in an unbuffered solution of I = O or 0.5 M NaCl. The acylation-rate constants, kappa 2, are: kappa H2O = 0.32 +/- 0.02 s-1 and kappa NaCl = 3.5 +/- 0.5 s-1, and they correlate to beta-trypsin (the most rapid single-chained form of the enzyme). The rate increasing effect of NaCl, together with the calculated electrostatic energies, indicate that the negative charge contribution of the Asp102-carboxylate to the stabilization of the imidazolium cation and tetrahedral oxyanion intermediate is larger of more orders, than that of the Cl- counter-ion, located in a less favourable position.
胰蛋白酶催化三联体中Asp102羧基的负电荷部分已被Cl-抗衡离子取代。在催化的酰化步骤中产生的His57咪唑鎓阳离子和Ser195四面体氧阴离子离子对通过Asp102的负电荷得以稳定。通过实验分析NaCl对胰蛋白酶酰化速率的影响以及静电势计算，研究了该负电荷正确定位的重要性。在pH 4.25±0.04的条件下，于I = 0或0.5 M NaCl的无缓冲溶液中，使用4-硝基苯基-4'-胍基苯甲酸酯活性位点滴定剂，采用停流技术在伪一级条件下测定酰化速率。酰化速率常数κ2分别为：κH2O = 0.32±0.02 s-1和κNaCl = 3.5±0.5 s-1，且它们与β-胰蛋白酶（该酶最快速的单链形式）相关。NaCl的速率增加效应以及计算出的静电能表明，Asp102羧基对咪唑鎓阳离子和四面体氧阴离子中间体稳定化的负电荷贡献比处于较不利位置的Cl-抗衡离子的贡献大多个数量级。
