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 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.
Angeli
Valeria
V
National Research Council-CNR, Inst. for Chemico-Physical Processes, Laboratory of Instrumental Analytical Chemistry, Via G. Moruzzi 1-56124 Pisa, Italy.
Tacito
Alessia
A
Paolicchi
Aldo
A
Barsacchi
Renata
R
Franzini
Maria
M
Baldassini
Riccardo
R
Vecoli
Cecilia
C
Pompella
Alfonso
A
Bramanti
Emilia
E
D000818:Animals; D002417:Cattle; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D010940:Plant Proteins / Q000378:metabolism; D010944:Plants / Q000201:enzymology; D026422:S-Nitrosoglutathione / Q000378:metabolism*; D012996:Solutions; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D005723:gamma-Glutamyltransferase / Q000378:metabolism*
D010940:Plant Proteins; D012996:Solutions; D026422:S-Nitrosoglutathione; D005723:gamma-Glutamyltransferase