Identification of a tyrosine residue in rat guanidinoacetate methyltransferase that is photolabeled with S-adenosyl-L-methionine.
Exposure of rat guanidinoacetate methyltransferase to ultraviolet light in the presence of S-adenosyl-L-[methyl-3H]methionine ([methyl-3H]AdoMet) results in covalent linking of radioactivity to the enzyme protein. The incorporation of radioactivity shows no lag and is linear with respect to time up to 1 h. The photolabeling is saturable with [methyl-3H]AdoMet, and the binding constant of the enzyme for AdoMet determined in this experiment is similar to that obtained by equilibrium dialysis. Low concentrations of competitive inhibitors S-adenosyl-L-homocysteine and sinefungin effectively prevent the photoinduced labeling by AdoMet. Although guanidinoacetate methyltransferase is irreversibly inactivated upon ultraviolet irradiation in the absence of AdoMet, the enzyme inactivated by 1-h exposure to ultraviolet irradiation has been shown to bind AdoMet with an affinity identical to that of the native enzyme. These results indicate that photolabeling occurs at the active site. Following proteolysis of the [methyl-3H]-AdoMet-labeled enzyme with chymotrypsin, a radioactive peptide is isolated having a sequence Asp-Thr-X-Pro-Leu-Ser-Glu-Glu-Thr-Trp. The peptide corresponds to residues 134-143, with X being modified Tyr-136. The same peptide is photolabeled when [carboxy-14C]AdoMet is used. High-performance liquid chromatography of this peptide after acid hydrolysis and phenyl isothiocyanate derivatization suggests that the entire molecule of AdoMet is attached to Tyr-136.
Takata
Y
Y
Department of Biochemistry, Toyama Medical and Pharmaceutical University Faculty of Medicine, Japan.
Fujioka
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D050937:Guanidinoacetate N-Methyltransferase; D066298:In Vitro Techniques; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010777:Photochemistry; D051381:Rats; D012436:S-Adenosylmethionine / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*; D014466:Ultraviolet Rays
D014443:Tyrosine; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C494508:Gamt protein, rat; D050937:Guanidinoacetate N-Methyltransferase