Understanding the Mechanism for Ribonucleotide Reductase Inactivation by 2'-...

Ribonucleotide reductase (RNR) is the key enzyme in the biosynthesis of deoxyribonucleotides. The enzyme has thus an attractive target for chemotherapies that fight proliferation-based diseases. 2'-Deoxy-2'methylenecytidine-5'-diphosphate (CH2dCDP) is a potent mechanism-based inhibitor of the enzyme RNR, which decomposes to an active alkylating furanone specie. The details of the inhibition mechanism are unknown, and experimental studies have indicated that some properties of the inactivation are dissimilar to those observed with a number of 2'-substituted 2'-deoxynucleotides mechanism-based inhibitors. To disclose the mechanism involved in RNR inactivation by CH2dCDP we explored the potential-energy surface in two different models of the system with different objectives in mind. In order to conveniently explore the reactional space, i.e. to study the possible reactions between the CH2dCDP and the RNR, we used a small model representing the active site of RNR with CH2dCDP using DFT. To provide further insights and efficiently account for the long-range RNR-CH2dCDP interactions and the stereochemical strain imposed by the protein scaffold we performed theoretical calculations on the more promising reactions using hybrid QM/MM calculations on a larger model system. We used quantum mechanics for the active-site region (CH2dCDP and active-site residues) and molecular mechanics for the surroundings (6373 atoms of the R1 monomer). The results obtained led us to understand the correct mechanism for RNR inactivation by CH2dCDP, and the furanone species formed presumably explains the dissimilarities observed with a number of 2'-substituted 2'-deoxynucleotides.