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 gamma-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 gamma-glutamyl moiety of GSNO to give S-nitroso-cysteinylglycine (CGNO) and gamma-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 K(m) of GGT for GSNO of 0.398+/-31 mM was thus found, comparable with K(m) values reported for other gamma-glutamyl substrates of GGT.
A kinetic study of gamma-glutamyltransferase (GGT)-mediated S-nitrosoglutathione catabolism
TACITO, ALESSIA;PAOLICCHI, ALDO;BARSACCHI, RENATA;FRANZINI, MARIA;VECOLI, CECILIA;POMPELLA, ALFONSO;
2009-01-01
Abstract
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 gamma-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 gamma-glutamyl moiety of GSNO to give S-nitroso-cysteinylglycine (CGNO) and gamma-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 K(m) of GGT for GSNO of 0.398+/-31 mM was thus found, comparable with K(m) values reported for other gamma-glutamyl substrates of GGT.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
