Gemcitabine 5‘-Triphosphate Is a Stoichiometric Mechanism-Based Inhibitor of Lactobacillus leichmannii Ribonucleoside Triphosphate Reductase:  Evidence for Thiyl Radical-Mediated Nucleotide Radical Formation

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 coupled to cob(II)alamin. From 22 to 255 ms the total spin concentration remained unchanged at 1.4 spins/RTPR, twice that predicted by the amount of cob(II)alamin determined by SF. However, with time the signal attributed to the thiyl radical-cob(II)alamin disappears and new signal(s) with broad feature(s) at g = 2.33 and a sharp feature at g = 2.00 appeared, suggesting formation of cob(II)alamin and a nucleotide-based radical with only dipolar interactions. These studies have been interpreted to support the proposal that an RTPR-based thiyl radical can give rise to a nucleotide-based radical.

Enhanced subunit interactions with gemcitabine-5′-diphosphate inhibit ribonucleotide reductases

“…Gemzar has two fluorines at C2Ј. Both are lost during inactivation, at least in the case of the E. coli and Lactobacillus leichmannii RNRs, which have thus far been examined (12,13). In addition, with [1Ј-3 H] F 2 CDP inactivation is accompanied by 0.5 eq of sugar labeling from F 2 C/␣, Ϸ20-40% of tyrosyl radical loss and 100% loss of RNR activity.…”

“…The monophosphate of F 2 C (F 2 CMP) is generated by deoxycytidine kinase (7) and is rapidly phosphorylated to the di-and triphosphates (F 2 CDP and F 2 CTP) (8,9). Diphosphorylated F 2 C (F 2 CDP) is an irreversible inhibitor of ribonucleotide reductase (RNR) (10)(11)(12)(13), and F 2 CTP functions as a chain terminator in the DNA polymerase reaction (14,15). Differentially phosphorylated states of gemzar can also interfere with other enzymes involved in nucleotide metabolism.…”

High-field EPR detection of a disulfide radical anion in the reduction of cytidine 5′-diphosphate by the E441Q R1 mutant of Escherichia coli ribonucleotide reductase

“…There is direct evidence that in the class II, adenosylcobalamin-dependent RNRs, the metallo-cofactor generates a thiyl radical in a kinetically-competent fashion (11,12). Recent studies with a class I RNR containing a diferric tyrosyl radical (Y⅐) cofactor and a class II RNR using cytidine nucleotide analogs provided direct evidence that the thiyl radical generates a 3Ј-nucleotide analog radical (13)(14)(15)(16). Once 1 is generated, there is also excellent model precedent that a ketyl radical 2 is generated subsequent to loss of water from the 2Ј position of the nucleotide (17,18).…”

Design and Synthesis of Novel Nucleoside Analogues: Oxidative and Reductive Approaches toward Synthesis of 2'-Fluoro Pyrimidine Nucleosides

“…23,24 By covalently binding to the active site, dFdCDP inhibits Ribonucleotide reductase (RNR), which catalyzes the reduction of ribonucleotides to deoxyribonucleotides. [25][26][27][28][29][30][31] The covalent inhibition of RNR causes decrease in the dNTP pool and consequently reduces dCTD activity. 32,33 In addition, decrease in the dNTP pool stimulates dFdC phosphorylation, thereby increasing the level of dFdCTP, making dFdCTP more likely to be incorporated into DNA competing with the NTPs.…”