Catalytic mechanism and inhibition of tRNA (uracil-5-)methyltransferase: evidence for covalent catalysis.
tRNA (Ura-5-)methyltransferase catalyzes the transfer of a methyl group from S-adenosylmethionine (AdoMet) to the 5-carbon of a specific Urd residue in tRNA. This results in stoichiometric release of tritium from [5-3H]Urd-labeled substrate tRNA isolated from methyltransferase-deficient Escherichia coli. The enzyme also catalyzes an AdoMet-independent exchange reaction between [5-3H]-Urd-labeled substrate tRNA and protons of water at a rate that is about 1% that of the normal methylation reaction, but with identical stoichiometry. S-Adenosylhomocysteine inhibits the rate of the exchange reaction by 2-3-fold, whereas an analogue having the sulfur of AdoMet replaced by nitrogen accelerates the exchange reaction 9-fold. In the presence (but not absence) of AdoMet, 5-fluorouracil-substituted tRNA (FUra-tRNA) leads to the first-order inactivation of the enzyme. This is accompanied by the formation of a stable covalent complex containing the enzyme, FUra-tRNA, and the methyl group of AdoMet. A mechanism for catalysis is proposed that explains both the 5-H exchange reaction and the inhibition by FUra-tRNA: the enzyme forms a covalent Michael adduct with substrate or inhibitor tRNA by attack of a nucleophilic group of the enzyme at carbon 6 of the pyrimidine residue to be modified. As a result, an anion equivalent is generated at carbon 5 that is sufficiently reactive to be methylated by AdoMet. Preliminary experiments and precedents suggest that the nucleophilic catalyst of the enzyme is a thiol group of cysteine. The potent irreversible inhibition by FUra-tRNA suggests that a mechanism for the "RNA" effects of FUra may also involve irreversible inhibition of RNA-modifying enzymes.
Santi
D V
DV
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Hardy
L W
LW
D000613:Aminobutyrates / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D005472:Fluorouracil / Q000494:pharmacology; D007700:Kinetics; D008745:Methylation; D012343:RNA, Transfer / Q000378:metabolism; D012435:S-Adenosylhomocysteine / Q000494:pharmacology; D012436:S-Adenosylmethionine / Q000494:pharmacology; D014316:Tritium; D012359:tRNA Methyltransferases / Q000037:antagonists & inhibitors*
D000613:Aminobutyrates; D014316:Tritium; C055412:N(4)-adenosyl-N(4)-methyl-2,4-diaminobutanoic acid; D012436:S-Adenosylmethionine; D012343:RNA, Transfer; D012435:S-Adenosylhomocysteine; D012359:tRNA Methyltransferases; C022769:tRNA(uracil-5)-methyltransferase; D005472:Fluorouracil