title: Reversible inactivation of recombinant rat liver guanidinoacetate methyltransferase by glutathione disulfide.
abstract: Recombinant rat liver guanidinoacetate methyltransferase is inactivated by glutathione disulfide (GSSG) following pseudo-first-order kinetics. A second-order rate constant of 20.8 M-1 min-1 is obtained at pH 7.5 and 30 degrees C. The inactivation is fully reversed by glutathione (GSH) in a pseudo-first-order fashion with a second-order rate constant of 11.1 M-1 min-1. The rate of inactivation is not affected by S-adenosylmethionine or guanidinoacetate, but complete protection against inactivation is observed in the presence of sinefungin plus guanidinoacetate. At equilibrium in the buffers containing various concentrations of GSH and GSSG, the enzyme shows activities that are dependent on the ratio but not on the total concentration of GSH and GSSG. A hyperbolic relationship is obtained between enzyme activity and [GSH]/[GSSG] ratio. The inactivation by GSSG is associated with the disappearance of approximately 1 mol of sulfhydryl group per mole of enzyme. These results indicate that inactivation of guanidinoacetate methyltransferase by GSSG is the consequence of the formation of a mixed disulfide between a protein thiol and glutathione. The equilibrium constant for the redox reaction, E-SH + GSSG in equilibrium with E-SSG + GSH, obtained from the equilibrium data (1.69) is in good agreement with the value determined as the ratio of second-order rate constants for reactivation and inactivation (1.87). The cysteine residue engaged in the mixed disulfide with glutathione is identified as Cys-15 by peptide analysis after consecutive treatment of the GSSG-inactivated enzyme with N-ethylmaleimide, 2-mercaptoethanol, and [14C]iodoacetate. The GSSG-inactivated enzyme binds S-adenosyl-methionine but not guanidinoacetate in the presence and absence of sinefungin. Native guanidinoacetate methyltransferase binds guanidinoacetate in the presence of sinefungin. The low overall redox equilibrium constant of 1.7-1.9 found for the reaction between guanidinoacetate methyltransferase and GSSG suggests that the activity of the enzyme is not amenable to modulation by the change in intracellular [GSH]/[GSSG] ratio.
authors.affiliation: Department of Biochemistry, Toyama Medical and Pharmaceutical University Faculty of Medicine, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Konishi
authors.forename: M
authors.initials: M
authors.lastname: Fujioka
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D003545:Cysteine / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004793:Enzyme Reactivators / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives* / Q000494:pharmacology; D019803:Glutathione Disulfide; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D050937:Guanidinoacetate N-Methyltransferase; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008623:Mercaptoethanol / Q000494:pharmacology; D008780:Methyltransferases / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism; D013057:Spectrum Analysis; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D004793:Enzyme Reactivators; D007461:Iodoacetates; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D008623:Mercaptoethanol; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C494508:Gamt protein, rat; D050937:Guanidinoacetate N-Methyltransferase; D005978:Glutathione; C004946:glycocyamine; D000241:Adenosine; D003545:Cysteine; D005033:Ethylmaleimide; D005998:Glycine; D019803:Glutathione Disulfide; C006235:sinefungin; D019807:Iodoacetic Acid