Escherichia coli dimethylallyl diphosphate : tRNA dimethylallyltransferase: pre-steady-state kinetic studies

Escherichia coil dimethylallyl diphosphate:tRNA dimethylallyltransferase (D MAPP-tRNA transferase) catalyzes the first step in the biosynthesis of the hypermodified A37 residue in tRNAs that read codons beginning with uridine. The mechanism of the enzyme-catalyzed reaction was studied by isotope trap ping, pre-steady-state rapid quench, and single turnover experiments. Isoto pe trapping indicated that the enzyme tRNA complex is catalytically compete nt, whereas the enzyme.DMAPP complex is not. The results are consistent wit h an ordered sequential mechanism for substrate binding where tRNA binds fi rst. The association and dissociation rate constants for the enzyme tRNA bi nary complex are 1.15 +/- 0.33 x 10(7) M-1 s(-1) and 0.06 +/- 0.01 s(-1), r espectively. Addition of DMAPP gives an enzyme.tRNA.DMAPP ternary complex i n rapid equilibrium with the binary complex and DMAPP. Rapid quench studies yielded a linear profile (k(cat) = 0.36 +/- 0.01 s(-1)) with no evidence f or buildup of enzyme-bound product. Product release from DMAPP-tRNA transfe rase is therefore not rate-limiting. The Michaelis constant for tRNA and th e equilibrium dissociation constant for DMAPP calculated from the individua l rate constants determined here are consistent with values obtained from a steady-state kinetic analysis. (C) 2000 Elsevier Science B.V. All rights r eserved.