GEMCITABINE 5'-TRIPHOSPHATE IS A STOICHIOMETRIC MECHANISM-BASED INHIBITOR OF LACTOBACILLUS-LEICHMANNII RIBONUCLEOSIDE TRIPHOSPHATE REDUCTASE - EVIDENCE FOR THIYL RADICAL-MEDIATED NUCLEOTIDE RADICAL FORMATION

Ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichm annii utilizes adenosylcobalamin and catalyzes the conversion of nucle oside triphosphates to deoxynucleoside triphosphates, One equivalent o f 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate, F(2)dCTP, rapidly i nactivates RTPR. Analysis of the reaction products reveals that inacti vation is accompanied by release of two fluoride ions and 0.84 equiv o f 5'-deoxyadenosine and attachment of 1 equiv of corrin covalently to an active-site cysteine residue of RTPR. No cytosine release was detec ted. Proteolysis of corrin-labeled RTPR with endoproteinase Glu-C and peptide mapping at pH 5.8 revealed that C419 was predominantly modifie d. The kinetics of the inactivation have been examined by stopped-flow (SF) UV-vis spectroscopy and rapid freeze quench (RFQ) electron param agnetic resonance (EPR) spectroscopy. Monitoring Delta A(525) nm shows that cob(II)alamin is formed with an apparent k(obs) of 50 s(-1), onl y 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 ti me points the EPR spectrum resembled a thiyl radical exchange coupled to cob(II)alamin. From 22 to 255 ms the total spin concentration remai ned unchanged at 1.4 spins/RTPR, twice that predicted by the amount of cob(II)alamin determined by SF. However, with time the signal attribu ted to the thiyl radical-cob(rr)alamin disappears and new signal(s) wi th broad feature(s) at g = 2.33 and a sharp feature at g = 2.00 appear ed, suggesting formation of cob(II)alamin and a nucleotide-based radic al with only dipolar interactions. These studies have been interpreted to support the proposal that an RTPR-based thiyl radical can give ris e to a nucleotide-based radical.