Recombinant rat liver guanidinoacetate methyltransferase: reactivity and function of sulfhydryl groups.
Rat liver guanidinoacetate methyltransferase, produced in Escherichia coli by recombinant DNA technique, possesses five cysteine residues per molecule. No disulfide bond is present. Analysis of the chymotryptic peptides derived from the iodo[14C]acetate-modified enzyme shows that Cys-90, Cys-15, Cys-219, and Cys-207 are alkylated by the reagent in order of decreasing reactivity. Incubation of the enzyme with excess 5,5'-dithiobis(2-nitrobenzoate) (DTNB) in the absence and presence of cystamine [2,2'-dithiobis(ethylamine)] causes the appearance of 4 and 5 mol of 2-nitro-5-mercaptobenzoate/mol of enzyme, respectively. Reaction of the methyltransferase with an equimolar amount of DTNB results in an almost quantitative disulfide cross-linking of Cys-15 and Cys-90 with loss of a large portion of the activity. The methyltransferase is completely inactivated by iodoacetate following nonlinear kinetics. Comparison of the extent of inactivation with that of modification of cysteine residues and the experiment with the enzyme whose Cys-15 and Cys-90 are cross-linked suggest that alkylation of Cys-15 and Cys-90 results in a partially active enzyme and that carboxymethylation of Cys-219 completely eliminates enzyme activity. The inactivation of guanidinoacetate methyltransferase by iodoacetate or DTNB is not protected by substrates. Furthermore, disulfide cross-linking of Cys-15 and Cys-90 or carboxymethylation of Cys-219 does not impair the enzyme's capacity to bind S-adenosylmethionine. Thus, these cysteine residues appear to occur outside the active-site region, but their integrity is crucial for the expression of enzyme activity.
Fujioka
M
M
Department of Biochemistry, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Japan.
Konishi
K
K
Takata
Y
Y
D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D050937:Guanidinoacetate N-Methyltransferase; D066298:In Vitro Techniques; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats
D004220:Disulfides; D007461:Iodoacetates; D004228:Dithionitrobenzoic Acid; D008780:Methyltransferases; C494508:Gamt protein, rat; D050937:Guanidinoacetate N-Methyltransferase; D003545:Cysteine; D019807:Iodoacetic Acid