Insight into the Mechanism of Inactivation of Ribonucleotide Reductase by Gemcitabine 5′-Diphosphate in the Presence or Absence of Reductant

Abstract

Gemcitabine 5′-diphosphate (F 2 CDP) is a potent inhibitor of ribonucleotide reductases (RNRs), enzymes that convert nucleotides (NDPs) to deoxynucleotides and are essential for DNA replication and repair. The E. coli RNR, an α2β2 complex, when incubated with one equivalent of F 2 CDP catalyzes the release of two fluorides and cytosine concomitant with enzyme inactivation. In the presence of reductant (thioredoxin/thioredoxin reductase/NADPH or DTT), the enzyme inactivation results from its covalent labeling of α with the sugar of F 2 CDP (one-label/α2β2). SDS PAGE analysis of the inactivated RNR without boiling of the sample reveals that α migrates as an 87 kDa and 110 kDa protein in a ratio of 0.6:0.4. When the reductant is omitted, RNR is inactivated by loss of the essential tyrosyl radical and formation of a new radical. Inactivation studies with C225S-α in the presence or absence of reductants, reveal it behaves like wt-RNR in the absence of reductant. Inactivated C225S-α migrates as an 87 kDa protein and is not covalently modified. C225 is one of the cysteines in RNR's active site that supplies reducing equivalents to make dNDPs. To identify the new radical formed, [1′-2 H] F 2 CDP was studied with wt-and C225S-RNR by 9 and 140 GHz EPR spectroscopy. These studies revealed that the new radical is nucleotide derived with g values of g x 2.00738, g y 2.00592, g z 2.00230 and with altered hyperfine interactions (apparent triplet collapsed to a doublet) relative to [1′-1 H] F 2 CDP. The EPR features are very similar to those we recently reported for the nucleotide radical generated with CDP and E441Q-RNR.

Strain Promoted Click Chemistry of 8-Azidopurine and 5-Azidopyrimidine Nucleosides and Nucleotides with Cyclooctynes and Applications to Living Cell Imaging

“…[286][287] Traditionally, kinases used for phosphorylation are typically specific for their natural substrates;…”

Biomimetic Modeling of the Nitrogen-centered Radical Postulated to occur during the Inhibition of Ribonucleotide Reductases by 2'-Azido-2'-deoxynucleotides.

“…The EPR characteristics of this new readical species shared many similarities with the semidione 9 generated when E441Q-RNR reacting with CDP. 26,47,52 A new dual-mechanism was proposed, as shown in Figure 13. 52,53 Stubbe proposed that following H3' abstraction and elimination of HF, the 3'-fluoroketyl radical 32 is formed, which is in resonance with the fluoro enone form 40.…”

“…26,47,52 A new dual-mechanism was proposed, as shown in Figure 13. 52,53 Stubbe proposed that following H3' abstraction and elimination of HF, the 3'-fluoroketyl radical 32 is formed, which is in resonance with the fluoro enone form 40. Addition of water to 40 and elimination of fluoride ion led to radical 41 without reductants, with EPR characteristics similar to semidione 9 (see Figure 5).…”

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.…”