title: Nuclear magnetic resonance study of the thioltransferase-catalyzed glutathione/glutathione disulfide interchange reaction.
abstract: The kinetics of the thioltransferase-catalyzed symmetrical glutathione/glutathione disulfide (GSH/GSSG) interchange reaction have been studied by 1H-nuclear magnetic resonance spectroscopy. Kinetic parameters were determined by analysis of exchange-broadened multiplet patterns and by the inversion-magnetization transfer method using concentrations of GSH, GSSG and pig liver thioltransferase similar to intracellular concentrations. The rate constant for the reaction of GSSG with thioltransferase to form a thioltransferase-glutathione mixed disulfide and GSH was estimated to be > or = 7.1(+/- 0.4).10(5) M-1 s-1. This reaction is proposed to be the first step in the mechanism by which the activity of some proteins is modulated by the thioltransferase-catalyzed formation of protein-glutathione mixed disulfides. The rate constant for the reaction of GSSG with thioltransferase is 4-5 orders of magnitude larger than rate constants for the analogous reaction of the thiolate groups of a variety of small molecules with GSSG. The symmetrical gamma-L-glutamyl-L-cysteine/gamma-L-glutamyl-L-cysteine disulfide (GCSH/GCSSCG), L-cysteinyl-glycine/L-cysteinyl-glycine disulfide (CGSH/CGSSGC) and cysteine/cystine (CSH/CSSC) thiol/disulfide interchange reactions were also studied as models for the GSH/GSSG interchange reaction. The GCSH/GCSSCG interchange reaction was found to be catalyzed by thioltransferase, and the rate constant for the reaction of GCSSCG with thioltransferase was estimated to be > or = 5.7(+/- 1.7).10(4) M-1 s-1. In contrast, the CGSH/CGSSGC and CSH/CSSC interchange reactions were found to be slow on the NMR time-scale for the conditions used in this research, both in the absence and presence of thioltransferase. The results suggest that the gamma-L-glutamyl-L-cysteinyl moiety of GSSG and of GSH-containing mixed disulfides is essential for their recognition by thioltransferase.
authors.affiliation: Department of Chemistry, University of California, Riverside 92521, USA.
authors.forename: D L
authors.initials: DL
authors.lastname: Rabenstein
authors.forename: K K
authors.initials: KK
authors.lastname: Millis
mesh_terms: D000818:Animals; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D054477:Glutaredoxins; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry*; D019803:Glutathione Disulfide; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D010088:Oxidoreductases / Q000378:metabolism*; D011490:Protein Disulfide Reductase (Glutathione); D013379:Substrate Specificity; D013552:Swine
chemical_list: D054477:Glutaredoxins; D003553:Cystine; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione); D005978:Glutathione; D003545:Cysteine; D019803:Glutathione Disulfide