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 (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 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 F(2)CDP is a substoichiometric inactivator of RNR. Incubation of this alpha/beta' with [1'-(3)H]-F(2)CDP or [5-(3)H]-F(2)CDP 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 alpha2. 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'-(3)H]-F(2)CDP 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 alpha6beta'6. A mechanism of inactivation common with halpha/beta is presented.

Insight into the mechanism of inactivation of ribonucleotide reductase by gemcitabine 5'-diphosphate in the presence or absence of reductant.

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 Escherichia coli RNR, an alpha2beta2 complex, when incubated with 1 equiv 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 alpha with the sugar of F(2)CDP (one label/alpha2beta2). SDS-PAGE analysis of the inactivated RNR without boiling of the sample reveals that alpha migrates as an 87 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-alpha in the presence or absence of reductants, reveal it behaves like wt-RNR in the absence of reductant. Inactivated C225S-alpha 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 a nucleotide derived with g values of g(x) 2.00738, g(y) 2.00592, and 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.