J. Biol. Chem., Vol. 261, Issue 14, 6346-6351, 05, 1986
Reaction of rat liver glycine methyltransferase with 5'-p- fluorosulfonylbenzoyladenosine
M Fujioka and Y Ishiguro
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.
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