NICKED MULTIFUNCTIONAL LOOP OF GLUTATHIONE SYNTHETASE STILL PROTECTS THE CATALYTIC INTERMEDIATE

A derivative of glutathione synthetase (GSHase) with the multifunction al loop cleaved (nicked GSHase) was compared to both a deletion mutant of the loop (loopless GSHase) and wild-type with the intact loop (wil d-type GSHase). The loop had been shown to be in a closed state in ord er to protect a catalytic intermediate and accelerate the reaction. Da ta 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, a nd also catalytic efficiency. The nicked loop did not accelerate the r eaction as fast as the intact loop; however, the catalytic intermediat e was protected from hydrolysis by the cleaved loop as effectively as by the intact loop. These results suggest that the fragmental loop ass umed the closed state. High concentrations of ATP showed some inhibito ry effects on wild-type GSHase, while both nicked and loopless GSHase were not inhibited, indicating that the fragments of the nicked loop f unctioned 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 accel erate glutathione synthesis. (C) 1997 Academic Press.