Mechanism of Inactivation of Human Ribonucleotide Reductase with p53R2 by Gemcitabine 5′-Diphosphate

Abstract

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside 5′-diphosphates to the corresponding deoxynucleotides supplying the dNTPs required for DNA replication and DNA repair. Class I RNRs require two subunits, α and β, for activity. Humans possess two β subunits: one involved in S phase DNA replication (β) and a second in mitochondrial DNA replication (β′ or p53R2) and potentially DNA repair. Gemcitabine (F 2 C) is used clinically as an anticancer agent and its phosphorylated metabolites target many enzymes involved in nucleotide metabolism, including RNR. The present investigation with α (specific activity of 400 nmol/min/mg) and β′ (0.6 Y•/β′2 and a specific activity of 420 nmol/min/mg) establishes F 2 CDP is a substoichiometric inactivator of RNR. Incubation of this α/β′ with [1′-3 H] F 2 CDP or [5-3 H] F 2 CDP and re-isolation of the protein by Sephadex G50 chromatography resulted in recovery 0.5 eq. of covalently bound sugar and 0.03 eq. of tightly associated cytosine to α2. SDS PAGE analysis (loaded without boiling) of the inactivated RNR, showed that 60% of α migrates as a 90 kDa protein and 40% as a 120 kDa protein. Incubation of [1′-3 H] F 2 CDP with active site mutants C444S/A, C218S/A, E431Q/D-α and the C-terminal tail C787S/A and C790S /A mutants, reveals that no sugar label is bound to the active site mutants of α and that in the case of C218S-α, α migrates as a 90 kDa protein. Analysis of the inactivated wt-α/β′ RNR by size exclusion chromatography indicates a quaternary structure of α6β′6. A mechanism of inactivation common with hα/β is presented.

Diversity in Overall Activity Regulation of Ribonucleotide Reductase

“…The dATP-inhibited complex binds the ␤ 2 subunit in the center of the ␣ 6 ring in such a way that the electron transport chain between the ␤ and ␣ subunits is disrupted. Less is known about the ATP-induced ␣ 6 complex, which has not been crystallized, but mutagenesis studies have indicated that it is structurally different from the inactive complex (9) and gel filtration analysis shows that it might bind up to three ␤ 2 subunits (12).…”

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

“…47,53,54 The detection and elucidation of this new radical species not only provided information for the mechanism of inhibition of RNR by Gemcitabine, but also suggested of a new intermediate for the mechanism of the natural substrate. …”

“…28,41,45,46 Gemcitabine, 2',2'-dideoxydifluorocytidine, is currently being clinically used as a drug for the treatment of nonsmall cell lung carcinomas and advanced pancreatic cancer. 37,47 It was first synthesized and demonstrated to have antitumor activity by Hertel and coworkers in 1990. 48 The mode of action of Gemcitabine is cell phase specific, meaning that it kills cells undergoing DNA synthesis.…”

“…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.…”

“…The EPR lineshapes and the radical adduct lifetimes also are individual characteristics that distinguish the nature of the trapped thiyl radical. 47 5,5-Dimethyl-1-pyrolline-N-oxide is probably the most successful spin trap for thiyl radicals since EPR spectra of DMPO-cysteinyl, DMPO-gluthionyl and DMPOpenicillamine adducts 45 are easily differentiated. 78 DEMPMO is a phosphorylated spin trap that reacts with thiyl radicals to produce EPR spectra from two diastereomers (46- …”