A kinetic study of gamma-glutamyltransferase (GGT)-mediated S-nitrosoglutathione catabolism.

S-Nitrosoglutathione (GSNO) is a nitric oxide (NO) donor compound which has been postulated to be involved in transport of NO in vivo. It is known that c-glutamyl transpeptidase (GGT) is one of the enzymes involved in the enzyme-mediated decomposition of GSNO, but no kinetics studies of the reaction GSNO-GGT are reported in literature. In this study we directly investigated the kinetics of GGT with respect to GSNO as a substrate and glycyl- glycine (GG) as acceptor co-substrate by spectrophotometry at 334 nm. GGT hydrolyses the c-glutamyl moiety of GSNO to give S-nitroso-cysteinylglycine (CGNO) and c-glutamyl-GG. However, as both the substrate GSNO and the first product CGNO absorb at 334 nm, we optimized an ancillary reaction coupled to the enzymatic reaction, based on the copper-mediated decomposition of CGNO yielding oxidized cysteinyl-glycine and NO. The ancillary reaction allowed us to study directly the GSNO/GGT kinetics by following the decrease of the characteristic absorbance of nitrosothiols at 334 nm. A Km of GGT for GSNO of 0.398 ± 31 mM was thus found, comparable with Km values reported for other c-glutamyl substrates of GGT.