title: tRNA-guanine transglycosylase from E. coli: a ping-pong kinetic mechanism is consistent with nucleophilic catalysis.
abstract: tRNA-guanine transglycosylase (TGT) is a key enzyme in the post-transcriptional modification of certain tRNAs with the pyrrolopyrimidine base queuine. TGT is required for pathogenicity in Shigella flexneri, a human pathogen, and therefore is potentially a novel antibacterial target. Previous work has indicated that the TGT reaction proceeds through a covalent enzyme-tRNA complex [Biochemistry 40 (2001) 14123]. To further substantiate this mechanism, the determination of the kinetic mechanism for the TGT reaction was undertaken. Computational and graphical analyses of initial velocity data are most consistent with a ping-pong kinetic mechanism. The modes of inhibition of 7-methylguanine with respect to both guanine (competitive) and tRNA (uncompetitive) indicate that tRNA binds first to the enzyme. This kinetic mechanism is consistent with the covalent intermediate chemical mechanism and with our earlier study of a mechanism-based inhibitor [7-fluoromethyl-7-deazaguanine, Biochemistry 34 (1995) 15539] in which TGT inactivation was dependent upon the presence of tRNA.
authors.affiliation: Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109-1065, USA.
authors.forename: DeeAnne M
authors.initials: DM
authors.lastname: Goodenough-Lashua
authors.forename: George A
authors.initials: GA
authors.lastname: Garcia
mesh_terms: D002384:Catalysis / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D015394:Molecular Structure; D010430:Pentosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D006147:Guanine; C008450:7-methylguanine; D010430:Pentosyltransferases; C020837:queuine tRNA-ribosyltransferase