ELECTROSTATIC EFFECTS IN TRYPSIN REACTIONS - INFLUENCE OF SALTS

The influence of inorganic salts on trypsin-catalyzed reactions has be en studied. It is shown that: (a) monovalent cations are reversible co mpetitive inhibitors of tryptic hydrolysis of cationic substrates, whe reas their binding has no effect on the reaction of neutral substrates ; (b) a nonelectrostatic salt effect on the binding of both cationic a nd non-ionic substrates is caused by changes in the thermodynamic acti vity coefficient of the substrate; (c) the rate of trypsin active-site acylation is not affected by inorganic salts with monovalent cations. The data suggest that low-molecular-mass substrates are extracted int o the enzyme microphase during substrate binding and further chemical transformations proceed without an access from surrounding medium. It is proposed that formation of a properly oriented dipole in the trypsi n binding pocket by the cationic group of the substrate and Asp189 car boxyl is responsible for the elevated acylation rate of trypsin active site by substrates containing lysine and arginine. Introduction of ad ditional negative charges into the enzyme molecule by chemical modific ation of lysyl residues by pyromellitic anhydride increased the specif icity of trypsin towards cationic substrates and inhibitors. Lysine re sidues are therefore considered as suitable targets for site-directed mutagenesis aimed at the improvement of selectivity and catalytic prop erties of trypsin.