Reaction of rat liver glycine methyltransferase with 5'-p-fluorosulfonylbenzoyladenosine.

Fujioka, M.; Ishiguro, Y

doi:10.1016/s0021-9258(19)84569-6

Cited by 21 publications

(10 citation statements)

References 22 publications

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“…Since AdoMet is known to bind to the free enzyme (Figure 5 and Fujioka & Ishiguro, 1986), the rate enhancement of phenylglyoxal inactivation in the presence of AdoMet suggests that the binding of AdoMet induces a conformational change of the enzyme so as to make the pertinent arginine more susceptible to modification. The finding that the inactivation is protected by acetate, a competitive inhibitor with respect to glycine, only in the presence of AdoMet also suggests that the conformational change caused by the binding of AdoMet generates the binding site for glycine.…”

Section: Discussionmentioning

confidence: 99%

“…However, almost nothing is known about which residues in the active site participate in binding and in catalysis. Thus far, the only amino acid residue that is thought to occur at or near the active site is a tyrosine (Fujioka & Ishiguro, 1986), but its functional role remains to be elucidated. In a number of proteins, arginine residues are found to be engaged in the binding of carboxyl or phosphoryl groups of substrates or cofactors (Riordan et al, 1977;Schneider, 1978).…”

mentioning

confidence: 99%

“…The amino acid calibration mixture was from Wako Pure Chemical Industries, Osaka, Japan. AdoMet was purified by C18 reverse-phase chromatography before use as described previously (Fujioka & Ishiguro, 1986). Other chemicals were of the highest purity available from commercial sources and were used without further purification.…”

mentioning

confidence: 99%

“…The extent of inactivation was monitored by measuring the residual enzyme activity. The enzyme activity was determined by coupling the methyltransferase reaction with S'-adenosylhomocysteinase and adenosine deaminase (Fujioka & Ishiguro, 1986). An aliquot (5-10 ^L) of the reaction mixture was added to 2.0 mL of the assay mixture containing 0.12 mM AdoMet, 2.0 mM glycine, and sufficient amounts of S'-adenosylhomocysteinase and adenosine deaminase in 20 mM potassium phosphate buffer, pH 7.5, and the decrease in absorbance at 265 nm due to the conversion of adenosine to inosine was followed.…”

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confidence: 99%

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Chemical modification of a functional arginine residue of rat liver glycine methyltransferase

Kawai¹,

Fujioka²

1987

Biochemistry

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Rat liver glycine methyltransferase is inactivated irreversibly by phenylglyoxal in potassium phosphate buffer. The inactivation obeys pseudo-first-order kinetics, and the apparent first-order rate constant for inactivation is linearly related to the reagent concentration. A second-order rate constant of 10.54 +/- 0.44 M-1 min-1 is obtained at pH 8.2 and 25 degrees C. Amino acid analysis shows that only arginine is modified upon treatment with phenylglyoxal. Sodium acetate, a competitive inhibitor with respect to glycine, affords complete protection in the presence of S-adenosylmethionine. Acetate alone has no effect on the rate of inactivation. The value of the dissociation constant for acetate determined from the protection experiment is in good agreement with that obtained by kinetic analysis. Comparison of the amount of [14C]phenylglyoxal incorporated into the protein and the number of arginine residues modified in the presence and absence of protecting ligands indicates that modification of one arginine residue per enzyme subunit eliminates the enzyme activity, and this residue is identified as Arg-175 by peptide analysis. The arginine-modified glycine methyltransferase appears to bind S-adenosylmethionine as the native enzyme does, as seen from quenching of the protein fluorescence by S-adenosylmethionine. These results suggest the requirement of Arg-175 in binding the carboxyl group of the substrate glycine.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Chemical modification of a functional arginine residue of rat liver glycine methyltransferase

Kawai¹,

Fujioka²

1987

Biochemistry

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“…AdoMet (grade II) obtained from Sigma Chemical Co. was purified before use by reverse-phase chromatography as described previously (Fujioka & Ishiguro, 1986), and its concentration was determined spectrophotometrically by using a value of «260 = 15.4 X 103 M'1 cm-1. Iodoacetic acid (Merck)…”

Section: Methodsmentioning

confidence: 99%

Recombinant rat liver guanidinoacetate methyltransferase: reactivity and function of sulfhydryl groups

Fujioka¹,

Kawai²,

Takata³

1988

Biochemistry

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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.

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Glycine N-methyltransferase

吳琰奇¹,

Wu²

Springer Handbook of Enzymes

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Reaction of rat liver glycine methyltransferase with 5'-p-fluorosulfonylbenzoyladenosine.

Cited by 21 publications

References 22 publications

Chemical modification of a functional arginine residue of rat liver glycine methyltransferase

Chemical modification of a functional arginine residue of rat liver glycine methyltransferase

Recombinant rat liver guanidinoacetate methyltransferase: reactivity and function of sulfhydryl groups

Glycine N-methyltransferase

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