Flexible loop that is novel catalytic machinery in a ligase. Atomic structure and function of the loopless glutathione synthetase.
The catalytic mechanism of glutathione synthetase is proposed to proceed via phosphorylation of the dipeptide substrate to yield an acyl phosphate intermediate; this intermediate is subsequently attacked by glycine, followed by loss of inorganic phosphate, leading to glutathione formation. A flexible loop (Ile226-Gly242) in Escherichia coli B glutathione synthetase is proposed to stabilize the acyl phosphate intermediate by preventing its decomposition by hydrolysis with water [Tanaka, T., Kato, H., Nishioka, T., & Oda, J. (1992) Biochemistry 31, 2259-2265; Tanaka, T., Yamaguchi, H., Kato, H., Nishioka, T., Katsube, Y., & Oda, J. (1993) Biochemistry 32, 12398-12404]. To investigate the function of the loop in the E. coli enzyme definitely, a loopless mutant in which the loop (Ile226-Arg241) was replaced with three residues of glycine was constructed. The crystal structure of the loopless mutant enzyme was essentially identical with that of the wild-type enzyme. Kinetic measurements showed that the replacement of the loop led to increases in the Km values, especially for the glycine, and a 930-fold decrease in the k0 value. Hence, the loopless mutant was 3 x 10(4) less active in terms of its specificity constant (k0/Km) for glycine than the wild-type enzyme. Moreover, the loopless mutant showed gamma-L-glutamyl-L-cysteine-dependent ATP hydrolase activity to almost the same extent as its glutathione synthetase activity. These studies support the fact that the loop enhances the recognition of glycine as well as stabilizes the acyl phosphate intermediate so that the intermediate rapidly reacts with glycine.
Kato
H
H
Institute for Chemical Research, Kyoto University, Japan.
Tanaka
T
T
Yamaguchi
H
H
Hara
T
T
Nishioka
T
T
Katsube
Y
Y
Oda
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D017931:DNA Primers; D004151:Dipeptides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D005981:Glutathione Synthase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity
D017931:DNA Primers; D004151:Dipeptides; D011994:Recombinant Proteins; D005981:Glutathione Synthase