Role of counter ions in trypsin acylation. NaCl effect

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.