MECHANISM-BASED INACTIVATION OF TRANSFER-RNA-GUANINE TRANSGLYCOSYLASEFROM ESCHERICHIA-COLI BY 2-AMINO-5-(FLUOROMETHYL)PYRROLO[2,3-D] PYRIMIDIN-4(3H)-ONE

In Escherichin coli, tRNA-guanine transglycosylase (TGT) catalyzes the incorporation of the queuine precursor preQ(1) -5-(aminomethyl)pyrrol o[2,3-d]pyrimidin-4(3H)-one] into tRNA. This precursor is further elab orated to queuine by two subsequent enzymic reactions [Slany, R. K., & Kersten, H. (1994) Biochimie 76, 1178-1182]. Our previous studies [Ho ops, G. C., Townsend, L. B., & Garcia, G. A. (1995) Biochemistry (in p ress)] 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 amin omethyl group of preQ(1)) at the 5 position. We report here that -5-(f luoromethyl)pyrrolo[2,3-d]pyrimidin-4(3H)-one (FMPP) inactivates TGT i n a time- and concentration-dependent manner with k(inact) = 0.074 min (-1) and K-I = 136 mu M. A competitive inhibitor (7-methyl-preQ(1)), w ith respect to preQ(1), of TGT [Hoops, G. C., Townsend, L. B., & Garci a, G. A. (1995) Biochemistry (in press)] protects the enzyme from inac tivation by FMPP. FMPP also acts as a competitive inhibitor (K-I = 114 mu M) 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 mu M FMPP, consistent with fluoride release pr eceding inactivation. FMPP appears to partition between ''normal'' tur nover (k(cat) = 0.461 min(-1) and K-m = 152 mu M), inactivation, and a n alternative processing to an unidentified, fluoride-released product . This inhibitor (FMPP), if it possesses suitable physical properties for cellular uptake, may serve as a useful biological probe in determi ning the role of the queuine modification in tRNA as well as an in vit ro mechanistic probe for the E. coli TGT.