Mechanism-based inactivation of tRNA-guanine transglycosylase from Escherichia coli by 2-amino-5-(fluoromethyl)pyrrolo[2,3-d]pyrimidin-4 (3H)-one

Biochemistry. 1995 Nov 28;34(47):15539-44. doi: 10.1021/bi00047a020.


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)

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Enzyme Inhibitors / metabolism*
  • Escherichia coli / enzymology*
  • Pentosyltransferases / antagonists & inhibitors
  • Pentosyltransferases / metabolism*
  • Pyrimidinones / metabolism*
  • Pyrroles / metabolism*
  • RNA, Transfer / metabolism*


  • 2-amino-5-(fluoromethyl)pyrrolo(2,3-d)pyrimidin-4(3H)-one
  • Enzyme Inhibitors
  • Pyrimidinones
  • Pyrroles
  • RNA, Transfer
  • Pentosyltransferases
  • queuine tRNA-ribosyltransferase