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 (F₂C) 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⁻¹ mg⁻¹) and β′ (0.6 Y·/β′2 and a specific activity of 420 nmol min⁻¹ mg⁻¹) establishes that F₂CDP is a substoichiometric inactivator of RNR. Incubation of this α/β′ with [1′-³H]-F₂CDP or [5-³H]-F₂CDP and reisolation of the protein by Sephadex G-50 chromatography resulted in recovery 0.5 equiv of covalently bound sugar and 0.03 equiv 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′-³H]-F₂CDP with active site mutants C444S/A, C218S/A, and 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.