Reaction of rat liver glycine methyltransferase with 5'-p-fluorosulfonylbenzoyladenosine.
Rat liver glycine methyltransferase is inactivated by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) in a pseudo-first order fashion at pH 7.5. The addition of dithiothreitol (20 mM) to the reaction mixture results in partial restoration of enzyme activity. A semilog plot of residual activity after dithiothreitol reactivation versus time is also linear, indicating that at least two essential residues are present on the enzyme and the modification of either of which causes total loss of activity. The inactivation is accompanied by incorporation of the radiolabel from adenine-labeled FSBA, but the amount of radioactivity fixed is not altered upon treatment with dithiothreitol. From this fact and the stoichiometry between the loss of dithiothreitol-sensitive activity and the number of sulfhydryl groups disappeared, it is suggested that the dithiothreitol-sensitive inactivation is the consequence of the FSBA-mediated formation of a disulfide between two sulfhydryl groups in close proximity. Although 4 mol of reagent are covalently bound per enzyme subunit, the kinetics of modification and inactivation show that the reaction at 1 residue, which is identified as tyrosine, is responsible for the dithiothreitol-insensitive inactivation. The substrate S-adenosylmethionine provides complete protection against both types of inactivation, but the dithiothreitol-insensitive inactivation is protected much more effectively with a Kd value comparable to the Km value. This suggests that the tyrosine is located at or near the active site of the methyltransferase.
Fujioka
M
M
Ishiguro
Y
Y
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004229:Dithiothreitol / Q000494:pharmacology; D050938:Glycine N-Methyltransferase; D007700:Kinetics; D008024:Ligands / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008433:Mathematics; D008780:Methyltransferases / Q000037:antagonists & inhibitors*; D051381:Rats; D012436:S-Adenosylmethionine / Q000378:metabolism
D008024:Ligands; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D012436:S-Adenosylmethionine; D008780:Methyltransferases; D050938:Glycine N-Methyltransferase; D000241:Adenosine; D004229:Dithiothreitol