Streptomyces griseus trypsin is stabilized against autolysis by the cooperation of a salt bridge and cation-pi interaction.
Streptomyces griseus trypsin (SGT) is a bacterial trypsin that lacks the conserved disulphide bond surrounding the autolysis loop. We investigated the molecular mechanism by which SGT is stabilized against autolysis. The autolysis loop connects to another surface loop via a salt bridge (Glu146-Arg222), and the Arg222 residue also forms a cation-pi interaction with Tyr217. Elimination of these bonds by site-directed mutagenesis showed that the surface salt bridge at Glu146-Arg222 is the main force stabilizing the enzyme against autolysis. The effect of the cation-pi interaction at Tyr217-Arg222 is small, however, its presence increases the half-life by about five hours and enhances the protein stability more than three-fold considering the catalytic activity in the presence of the salt bridge. The melting temperature also showed cooperation between the salt bridge and cation-pi interaction. These findings show that S. griseus trypsin is stabilized against autolysis through a cooperative network of a salt bridge and cation-pi interaction, which compensate for the absence of the conserved C136-C201 disulphide bond.
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Gusong-dong, Yusong-gu, Taejon 305-701, Korea.
Weon Sup
WS
Lee
Chi Hye
CH
Park
Si Myung
SM
Byun
D001120:Arginine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001433:Bacteriolysis / Q000235:genetics; D002412:Cations; D004795:Enzyme Stability / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000235:genetics; D013305:Streptomyces griseus / Q000201:enzymology* / Q000235:genetics; D014357:Trypsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D002412:Cations; D018698:Glutamic Acid; D001120:Arginine; D014357:Trypsin