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

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, alpha and beta, for activity. Humans possess two beta subunits: one involved in S phase DNA replication (beta) and a second in mitochondrial DNA replication (beta' 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 alpha (specific activity of 400 nmol min(-1) mg(-1)) and beta' (0.6 Y./beta'2 and a specific activity of 420 nmol min(-1) mg(-1)) establishes that F2CDP is a substoichiometric inactivator of RNR. Incubation of this alpha/beta' with [1'-H-3]-F2CDP or [5-H-3]-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 alpha 2. SDS-PAGE analysis (loaded without boiling) of the inactivated RNR showed that 60% of alpha migrates as a 90 kDa protein and 40% as a 120 kDa protein. Incubation of [1'-H-3]-F2CDP with active site mutants C444S/A, C218S/A, and E431Q/D-alpha and the C-terminal tail C787S/A and C790S/A mutants reveals that no sugar label is bound to the active site mutants of alpha and that, in the case of C218S-alpha, alpha migrates as a 90 kDa protein. Analysis of the inactivated wt-alpha/beta' RNR by size exclusion chromatography indicates a quaternary structure of alpha 6 beta'6. A mechanism of inactivation common with h alpha/beta is presented.