Abstract
The reactions catalyzed by y-glutamylcysteine syn-thetase and glutamine synthetase are thought to proceed via enzyme-bound y-glutamyl phosphate inter-mediates. We investigated the possibility that S-sulfo-cysteine and S-sulfohomocysteine might act as analogs of y-glutamyl phosphate or of the associated putative tetrahedral intermediates. The D-and L-enantiomers of S-sulfocysteine and S-sulfohomocysteine were found to rapidly inactivate rat kidney y-glutamylcysteine synthetase but to be reversible inhibitors of sheep brain glutamine synthetase. Inactivation of y-glutamylcys-teine synthetase does not require ATP and is associated with noncovalent binding of close to 1 mol of inacti-vator/mol of enzyme. The findings indicate that the S-sulfo amino acids are transition-state analogs, and that binding of S-sulfo amino acid to the enzyme induces formation of a very stable enzyme-inactivator complex. The data suggest that stabilization of the enzyme-inactivator complex results from interactions involving the sulfenyl sulfur atom of the S-sulfo amino acid and the active site thiol group of the enzyme. y-Glutamylcysteine synthetase and glutamine synthetase catalyze similar reactions in which enzyme-bound y-glutamyl phosphate (Fig. 1, I) is formed as an intermediate (reaction 1 and 2) (1,2): Enzyme + L-glutamate + ATP $ enzyme[y-Glu-PI [ADP] (1) (2) Enzyme[y-Glu-PI [ADP] + NHzR enzyme + 7-Glu-NHR + ADP + Pi In glutamine synthetase, R = H (NH3 may be replaced by In y-glutamylcysteine synthetase, R = HSCH,CHCOO-(cysteine may be replaced by a-aminobutyrate). (Both reactions require divalent metal ions (Mg2+, Mn2+)). The evidence for intermediate formation of enzyme-bound y-glutamyl phosphate in the glutamine synthetase reaction 2 includes the findings that this enzyme catalyzes (a) cyclization of D-and L-glutamate to 5-oxoproline (reaction 3 (3, 4)), (b)