Mechanism-Based Inactivation of tRNA-Guanine Transglycosylase from Escherichia coli by 2-Amino-5-(fluoromethyl)pyrrolo[2,3-d]pyrimidin-4(3H)-one

Abstract

In Escherichia coli, tRNA-guanine transglycosylase (TGT) catalyzes the incorporation of the queuine precursor preQ1 [2-amino-5-(aminomethyl)pyrrolo[2,3-d]pyrimidin-4(3H)-one] into tRNA. This precursor is further elaborated to queuine by two subsequent enzymic reactions [Slany, R. K., & Kersten, H. (1994) Biochimie 76, 1178-1182]. Our previous studies [Hoops, G. C., Townsend, L. B., & Garcia, G. A., (1995) Biochemistry (in press)] on a series of synthetic 5- and 6-substituted 2-aminopyrrolo[2,3-d]pyrimidin-4(3H) -ones have revealed that the E. coli TGT tolerates a wide diversity of substituents (isosteric, or nearly so, to the aminomethyl group of preQ1) at the 5 position. We report here that 2-amino-5-(fluoromethyl)pyrrolo[2,3-d]pyrimidin-4 (3H)-one (FMPP) inactivates TGT in a time- and concentration-dependent manner with k(inact) = 0.074 min-1 and KI = 136 microM. A competitive inhibitor (7-methyl-preQ1), with respect to preQ1, of TGT [Hoops, G.C., Townsend, L.B., & Garcia, G.A. (1995) Biochemistry (in press)] protects the enzyme from inactivation by FMPP. FMPP also acts as a competitive inhibitor (KI = 114 microM) of TGT under initial velocity conditions. The rate of fluoride release from FMPP is slightly faster (0.064 min-1) than the k(inact) (0.053 min-1) at 300 microM FMPP, consistent with fluoride release preceding inactivation. FMPP appears to partition between "normal" turnover (kcat = 0.461 min-1 and Km = 152 microM), inactivation, and an alternative processing to an unidentified, fluoride-released product.(ABSTRACT TRUNCATED AT 250 WORDS)

tRNA–guanine transglycosylase from E. coli: a ping-pong kinetic mechanism is consistent with nucleophilic catalysis

“…Although this rate of washout is low (slow) it does provide evidence that the first chemical step can occur in the absence of the second substrate. Finally, ping-pong kinetics are consistent with our earlier study of a mechanism-based inhibitor of TGT, 7-fluoromethyl-7-deazaguanine (FMPP) [38]. In those studies, we observed that FMPP inactivation of TGT was dependent upon tRNA and that FMPP is competitive with respect to guanine, consistent with FMPP interacting with a covalent TGTtRNA intermediate.…”

Transglycosylation: A mechanism for RNA modification (and editing?)

“…In addition to a thorough understanding of the kinetics of the TGT reaction, studies over the last decade have helped define the recognition of both the tRNA [22,[25][26][27][28][29][30][31][32][33] and heterocyclic base substrates [34][35][36] and have also helped unveil the roles that certain active-site amino acid residues serve in catalysis. Early work in our laboratory determined that TGT is a zinc metalloenzyme.…”

Queuine tRNA-ribosyltransferase

“…2 Source Organism <1> Escherichia coli [1,4,10,15,16,17,18,19,21,22,23,24] <2> Oryctolagus cuniculus [2,5,20] <3> Salmonella typhimurium [3] <4> Xenopus laevis [6,8] <5> Triticum aestivum [7] <6> Rattus norvegicus [9] <7> Mus musculus [11] <8> Homo sapiens [11,13] <9> Shigella flexneri [11,12,23] <10> Haemophilus influenza [11] <11> Helicobacter pylori [11] <12> Synechocystis sp. [11] <13> Thermotoga maritima [11] <14> Caenorhabditis elegans [11] <15> Methanococcus jannaschii [11] <16> Archaeoglobus fulgidus [11] <17> Methanobacterium thermoautotrophicum [11] <18> Zymomonas mobilis [14,24] <19> Pyrococcus horikoshii [15] 3 Reaction and Specificity…”

“…Substrates and products S 2-amino-5-(fluoromethyl)pyrrolo [2,3-d]pyrimidin-4(3H)-one + tRNAguanine <1> (<1>, 2-amino-5-(fluoromethyl)pyrrolo [2,3-d]pyrimidin-4(3H)one appears to partition between: 1. normal turnover, 2. inactivation, 3. an alternative processing to an unidentrified fluoride-released product [19]) (Reversibility: ? <1> [19]) [19] P guanine + tRNA2-amino-5-(fluoromethyl)pyrrolo [2,3-d]pyrimidin-4(3H)one S 2-thiohypoxanthine + tRNAguanine <6> (Reversibility: ? <6> [9]) [9] P guanine + tRNA2-thiohypoxanthine S 6-thioguanine + tRNAguanine <6> (Reversibility: ?…”

“…<2, 3, 6> [2,3,9]) [1, 2, 3, 5, 7, 9] P tRNAguanine + guanine S queuine + tRNAguanine <2, 4, 5, 6> (<4>, nucleosides in position 36, 37 and 38 influence the efficiency of conversion of G-34 to queuosine-34 [6]; <6>, tRNA Tyr , tRNA His , tRNA Asn , or tRNAASp from an E. coli mutant or rat ascites hepatoma cells [9]) (Reversibility: ir <2> [5]; ? <4, 5, 6> [6,7,9]) [5,6,7,9] P guanine + tRNAqueuine inactivation, 3. an alternative processing to an unidentrified fluoride-released product [19]) [19] 3-deazaguanine <2> (<2>, 0.25 mM, 50% inhibition [5]) [5] 6-methylmercaptoguanine <2> (<2>, 0.05 mM, 65% inhibition [5]) [5] 6-thioguanine <2> (<2>, 0.05 mM, 74% inhibition [5]) [5] 7-deazaguanine <2> (<2>, 0.06 mM, 27% inhibition [5]) [5] 7-methylguanine <1, 2> (<2>, 0.05 mM, 94% inhibition [5]) [5,23] 8-azaguanine <2> (<2>, 0.05 mM, complete inhibition [5]) [5] 8-bromoguanine <2> (<2>, 0.05 mM, 86% inhibition [5]) [5] 8-dimethylaminomethyl-7-deazaguanine <2> (<2>, 0.05 mM, 32% inhibition [5]) [5] Cd 2+ <5> (<5>, 10 mM [7]) [7] Co 2+ <5> (<5>, 10 mM [7]) [7] Cu 2+ <5> (<5>, 10 mM [7]) [7] Mn 2+ <5> (<5>, 10 mM [7]…”