Purification and characterization of transfer RNA (guanine-1)methyltransferase from Escherichia coli.
The tRNA modifying enzyme, tRNA (guanine-1)methyltransferase has been purified to near homogeneity from an overproducing Escherichia coli strain harboring a multicopy plasmid carrying the structural gene of the enzyme. The preparation gives a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is probably a single polypeptide chain of molecular weight 32,000. The amino acid composition is presented and the NH2-terminal amino acid sequence was established to be H2N-Met-Trp-Ile-Gly-Ile-Ile-Ser-Leu-Phe-Pro. The enzyme has a pI of 5.2. The tRNA (guanine-1)-methyltransferase has a pH optimum of 8.0-8.5, an apparent Km of 5 microM for S-adenosylmethionine. S-adenosylhomocysteine is a competitive inhibitor for the enzyme with an apparent Ki of 6 microM. Spermidine or putrescine are not required for activity, but they stimulate the rate of methylation 1.2-fold with optima at 2 and 6 mM, respectively. Ammonium ion is not required and is inhibitory at concentrations above 0.15 M. Magnesium ion inhibited the activity at a concentration as low as 2 mM. Sodium and potassium ions were inhibitory at concentrations above 0.1 M. The molecular activity of tRNA (guanine-1)-methyltransferase was calculated to 10.0 min-1. It was estimated that the enzyme is present at 80 molecules/genome in cells growing with a specific growth rate of 1.0.
D000595:Amino Acid Sequence; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005796:Genes; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D009154:Mutation; D010957:Plasmids; D012359:tRNA Methyltransferases / Q000235:genetics / Q000302:isolation & purification*
D012359:tRNA Methyltransferases; C036950:tRNA (guanine-N1-)-methyltransferase