Enhanced subunit interactions with gemcitabine-5'-diphosphate inhibit ribonucleotide reductases

Abstract

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The class I RNRs are composed of two subunits, alpha and beta, with proposed quaternary structures of alpha2beta2, alpha6beta2, or alpha6beta6, depending on the organism. The alpha subunits bind the nucleoside diphosphate substrates and the dNTP/ATP allosteric effectors that govern specificity and turnover. The beta2 subunit houses the diferric Y* (1 radical per beta2) cofactor that is required to initiate nucleotide reduction. 2',2'-difluoro-2'-deoxycytidine (F2C) is presently used clinically in a variety of cancer treatments and the 5'-diphosphorylated F2C (F2CDP) is a potent inhibitor of RNRs. The studies with [1'-(3)H]-F2CDP and [5-(3)H]-F2CDP have established that F2CDP is a substoichiometric mechanism based inhibitor (0.5 eq F2CDP/alpha) of both the Escherichia coli and the human RNRs in the presence of reductant. Inactivation is caused by covalent labeling of RNR by the sugar of F2CDP (0.5 eq/alpha) and is accompanied by release of 0.5 eq cytosine/alpha. Inactivation also results in loss of 40% of beta2 activity. Studies using size exclusion chromatography reveal that in the E. coli RNR, an alpha2beta2 tight complex is generated subsequent to enzyme inactivation by F2CDP, whereas in the human RNR, an alpha6beta6 tight complex is generated. Isolation of these complexes establishes that the weak interactions of the subunits in the absence of nucleotides are substantially increased in the presence of F2CDP and ATP. This information and the proposed asymmetry between the interactions of alphanbetan provide an explanation for complete inactivation of RNR with substoichiometric amounts of F2CDP.

Scheme 1.

Fig. 1.

Time-dependent inactivation of human RNR by F₂CDP. Inactivation mixture contained final concentrations of α, β, 1.2 μM; F₂CDP, 0.6 μM (■), and 6 μM (▴); ATP, 3 mM; and DTT, 5 mM. Aliquots were removed at various times and diluted 4-fold for determination of RNR activity. Control experiment (♦) is identical to the experiment except that F₂CDP was omitted.

Fig. 2.

SEC on a Superose 12 column to detect complex formation (α2β2) in E. coli RNR incubated with ATP in the presence or absence of [1′-³H] F₂CDP. Elution buffer contains 0.5 mM ATP. (A–C) Presence of F₂CDP. (A) Elution profile monitored by A_{280 nm} and scintillation counting (♦). (B) Fractions through the protein peak in A monitored by SDS/PAGE. Note the slower migrating band (5% of the protein) is an altered conformation of α. (C) Analysis of the ratio of α:β (−), using standard curves generated from known amounts of α and β. (D–F) Absence of F₂CDP. (D) Elution profile. (E) Fractions through the protein peak in D monitored by SDS/PAGE. (F) Analysis of the ratio of α:β, using standard curves generated from known amounts of α and β.

Fig. 3.

SEC on a Superdex 200 column to detect the complex formation (αnβn) in human RNR upon inactivation by F₂CDP/ATP. (A) The elution profile monitored by A₂₁₄ and scintillation counting (■). (B) Fractions through the protein peak in A monitored by SDS/PAGE and analysis of the ratio of α:β using standard curves generated from known amounts of α and β. Peak I contains α and β; Peak II contains Arna; and Peak III contains no α or β, but an unidentified protein of monomer molecular mass 65 kDa. We have repeated this experiment with homogeneous α (provided by the C. G. Dealwis laboratory, University of Tennessee, Knoxville, TN). The results are identical, but look cleaner, because the E. coli contaminating protein has been removed.