Biochemical studies on the stability of streptomyces griseus trypsin

Abstract

[Part I ] Streptomyces griseus trypsin (SGT) is a bacterial trypsin that lack the conserved disulphide bond surround the autolysis loop. We investigated the molecular mechanism by which SGT was stabilized against autolysis. The autolysis loop connected to another surface loop via salt bridge (Glu146-Arg222) and that the Arg222 residue could also form a cation-π interaction with Tyr217. Elimination of these bonds by site-directed mutagenesis showed that the surface salt bridge at Glu146-Arg222 was the main force in stabilizing enzyme against autolysis. The effect of cation-π interaction at Tyr217-Arg222 itself was little, however, it could make longer the half-life for about five hours and enhance the protein stability over three fold in considering the catalytic activity by the presence of the salt bridge. Melting temperature also showed the cooperation between the salt bridge and the cation-π interaction. These findings showed that S. griseus trypsin was stabilized against autolysis by forming a cooperative network of a salt bridge and a cation-π interaction, which could compensated for the absence of the conserved C136-C201 disulphide bond. [Part II] Streptomyces griseus trypsin (SGT) shows higher catalytic activity and stability than bovine trypsin. The activity and stability of SGT was investigated in aqueous ethanol for its potential use in organic solvent. Up to 80% ethanol, the catalytic activity of SGT was still higher than that of bovine trypsin measured in water. As ethanol concentration increased, SGT became more reactive to the Lys-containing substrate than Arg-containing substrate, which was resulted from the difference of their $K_m$ values. Calcium stabilized enzyme against autolysis in aqueous ethanol, but it can destabilize when the concentration of ethanol exceeded 80%, in that concentration enzyme lost its activity by the spontaneous denaturation. These phenomena can be explained by the effect of organic solvent to the conformational restrict...