Nicked multifunctional loop of glutathione synthetase still protects the catalytic intermediate.
A derivative of glutathione synthetase (GSHase) with the multifunctional loop cleaved (nicked GSHase) was compared to both a deletion mutant of the loop (loopless GSHase) and wild-type with the intact loop (wild-type GSHase). The loop had been shown to be in a closed state in order to protect a catalytic intermediate and accelerate the reaction. Data indicated that cleavage of the loop resulted in a drastic decrease in glutathione synthetic activity which was similar to the results for the loop deletion. Kinetic analyses indicated that the manipulations of the loop impaired the substrate affinity, especially for glycine, and also catalytic efficiency. The nicked loop did not accelerate the reaction as fast as the intact loop; however, the catalytic intermediate was protected from hydrolysis by the cleaved loop as effectively as by the intact loop. These results suggest that the fragmental loop assumed the closed state. High concentrations of ATP showed some inhibitory effects on wild-type GSHase, while both nicked and loopless GSHase were not inhibited, indicating that the fragments of the nicked loop functioned independently. In conclusion, it is postulated that the two fragments of the nicked loop independently assumed the closed state to protect the catalytic intermediate and have lost the ability to accelerate glutathione synthesis.
Tanaka
T
T
Institute for Chemical Research, Kyoto University, Japan.
Nishioka
T
T
Oda
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D005978:Glutathione / Q000378:metabolism; D005981:Glutathione Synthase / Q000737:chemistry*; D007700:Kinetics; D009038:Motion; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014357:Trypsin
D001426:Bacterial Proteins; D000255:Adenosine Triphosphate; D014357:Trypsin; D005981:Glutathione Synthase; D005978:Glutathione