Theoretical studies on the mode of inhibition of ribonucleotide reductase by 2'-substituted substrate analogues.

Several 2'-substituted-2'-deoxyribonucleotides are potent time-dependent inactivators of the enzyme ribonucleotide reductase (RNR), which function by destructing its essential tyrosil radical and/or by performing covalent addition to the enzyme. The former leads to inhibition of the R2 dimer of RNR and the latter to inhibition of the R1 dimer. Efforts to elucidate the mechanism of inhibition have been undertaken in the last decades, and a general mechanistic scheme has emerged. Accordingly, two alternative pathways lead either to the inhibition of R1 or R2, for which the 2'-chloro-2'-deoxynucleotides serve as the model for the inhibition of R1 and the 2'-azido-2'-deoxynucleotides the model for the inhibition of R2. However, the underlying reason for the different behavior of the inhibitors has remained unknown until now. Moreover, a fundamental mechanistic alternative has been proposed, based on results from biomimetic reactions, in which the 2'-substituents would be eliminated as radicals, and not as anions, as previously assumed. This would lead to further reactions not predicted by the existing mechanistic scheme. To gain a better understanding we have performed high-level theoretical calculations on the active site of RNR. Results from this work support the general Stubbe's paradigm, although some changes to that mechanism are necessary. In addition, a rational explanation of the factors that determine which of the dimers (R1 or R2) will be inactivated is provided for the first time. It has been demonstrated also that the 2'-substituents are indeed eliminated as anions, and not as radicals. Biomimetic experiments have led to different results because they lack a basic group capable of deprotonating the 3'-HO group of the substrate. It has been found here that the chemical character of the leaving group (radical or anionic) can be manipulated by controlling the protonation state of the 3'-HO group.