Mutational and proteolytic studies on a flexible loop in glutathione synthetase from Escherichia coli B: the loop and arginine 233 are critical for the catalytic reaction.
The function of the flexible loop which is disordered in crystal structure analysis of glutathione synthetase from Escherichia coli B has been investigated by limited proteolysis and kinetic measurements for the wild-type and mutant enzymes. Proteolysis of the intact enzyme using arginyl endopeptidase or trypsin brought about a time-dependent decrease in the enzymatic activity and the production of protein fragments. SDS-polyacrylamide gel electrophoresis and peptide sequence analysis showed that only a peptide bond between arginine 233 and glycine 234 in the loop was cleaved. Further, native polyacrylamide gel electrophoresis revealed that the cleaved enzyme retained almost the same quaternary structure as that of the wild-type enzyme. Upon protease treatment, the presence of substrates, ATP and/or gamma-L-glutamyl-L-cysteine (gamma-Glu-Cys), protected the loop from cleavage, but the presence of glycine was not capable of protecting it. In addition, replacement of arginine 233 in the loop with lysine by site-directed mutagenesis increased the Michaelis constants for gamma-Glu-Cys and glycine by factors of 28 and 213, respectively. The protection against cleavage on a similar protease incubation of this mutant enzyme was also observed in the presence of ATP and/or gamma-Glu-Cys, but the effect in the presence of both substrates was half as large as that for the wild-type enzyme. These results suggest that the loop covers the active site while ATP and gamma-Glu-Cys bind there and that it protects the unstable gamma-Glu-Cys phosphate intermediate from decomposition by bulk water.(ABSTRACT TRUNCATED AT 250 WORDS)
Tanaka
T
T
Institute for Chemical Research, Kyoto University, Japan.
Kato
H
H
Nishioka
T
T
Oda
J
J
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005981:Glutathione Synthase / Q000737:chemistry* / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012697:Serine Endopeptidases
D001120:Arginine; D012697:Serine Endopeptidases; C060325:arginine endopeptidase; D005981:Glutathione Synthase