Gemcitabine 5'-triphosphate is a stoichiometric mechanism-based inhibitor of Lactobacillus leichmannii ribonucleoside triphosphate reductase: evidence for thiyl radical-mediated nucleotide radical formation

Biochemistry
D J SilvaM J Robins

Abstract

Ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii utilizes adenosylcobalamin and catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates. One equivalent of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate, F2dCTP, rapidly inactivates RTPR. Analysis of the reaction products reveals that inactivation is accompanied by release of two fluoride ions and 0.84 equiv of 5'-deoxyadenosine and attachment of 1 equiv of corrin covalently to an active-site cysteine residue of RTPR. No cytosine release was detected. Proteolysis of corrin-labeled RTPR with endoproteinase Glu-C and peptide mapping at pH 5.8 revealed that C419 was predominantly modified. The kinetics of the inactivation have been examined by stopped-flow (SF) UV-vis spectroscopy and rapid freeze quench (RFQ) electron paramagnetic resonance (EPR) spectroscopy. Monitoring DeltaA525 nm shows that cob(II)alamin is formed with an apparent kobs of 50 s-1, only 2. 5-fold slower than a similar experiment carried out with cytidine 5'-triphosphate (CTP). The same reaction mixture was thus quenched at times from 22 ms to 30 s and examined by EPR spectroscopy. At early time points the EPR spectrum resembled a thiyl radical exchange cou...Continue Reading

References

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Apr 7, 1970·Biochimica Et Biophysica Acta·B G MalmströmT Vänngård

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Citations

Dec 16, 1998·Trends in Biochemical Sciences·J Stubbe, P Riggs-Gelasco
Dec 18, 2013·Journal of Medicinal Chemistry·Jesse PulidoStanislaw F Wnuk
Aug 4, 1999·Proceedings of the National Academy of Sciences of the United States of America·C C LawrenceJ Stubbe
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Feb 22, 2005·Biochimica Et Biophysica Acta·Catherine Berthomieu, Rainer Hienerwadel
Dec 6, 2017·Biotechnology and Bioengineering·Cheng DuZheng Jian Li

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