Stereochemical retention of the configuration in the action of Fhit on phosphorus-chiral substrates

Andreas Abend, Preston N. Garrison, Larry D. Barnes, Perry A. Frey

Research output: Contribution to journalArticlepeer-review

32 Scopus citations


Fhit is the protein product of FHIT, a candidate human tumor suppressor gene. Fhit catalyzes the hydrolysis of diadenosine triphosphate (Ap3A) to AMP and ADP. Fhit is here shown to catalyze the hydrolysis in H218O with production of adenosine 5'-[18O]phosphate and ADP, proving that the substitution of water is at Pα and not at Pβ. The chain fold of Fhit is similar to that of galactose-1-phosphate uridylyltransferase, which functions by a double-displacement mechanism through the formation of a covalent nucleotidyl - enzyme intermediate and overall retention of configuration at Pα. The active site of Fhit contains a histidine motif that is reminiscent of the HPH motif in galactose-1-phosphate uridylyltransferases, in which the first histidine residue serves as the nucleophilic catalyst to which the nucleotidyl group is bonded covalently in the covalent intermediate. In this work, the Fhit-catalyzed cleavage of (R(P))- and (S(P))-γ-(m-nitrobenzyl) adenosine 5'-O-1-thiotriphosphate (mNBATPαS) in H218O to adenosine 5'- [18O]thiophosphate is shown to proceed with overall retention of configuration at phosphorus. γ-(m-Nitrobenzyl) adenosine 5'-O-triphosphate (mNBATP) is approximately as good a substrate for Fhit as Ap3A, and both (R(P))- and (S(P))-mNBATPαS are substrates that react at about 0.5% of the rate of Ap3A. The stereochemical evidence indicates that hydrolysis by Fhit proceeds by a double-displacement mechanism, presumably through a covalent AMP - enzyme intermediate.

Original languageEnglish (US)
Pages (from-to)3668-3676
Number of pages9
Issue number12
StatePublished - Mar 23 1999

ASJC Scopus subject areas

  • Biochemistry


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