Theoretical studies on the mechanism of inhibition of Ribonucleotide Reductase by (E)-2'-Fluoromethylene-2'-deoxycitidine-5'-diphosphate.
(E)-2'-Fluoromethylene-2'-deoxycitidine-5'-diphosphate (FMCDP) is a potent time-dependent inactivator of the enzyme Ribonucleotide Reductase, which operates by destructing an essential tyrosil radical and performing a covalent addition to an active site residue. Considerable effort to elucidate the inhibition mechanism has been undertaken in recent years, and some insights have been obtained. Although a mechanistic proposal has been put forward, based on a general paradigm of inhibition of RNR by 2' substituted substrate analogues, details about the mechanism have remained elusive. Namely, the exact residue that adds to the inhibitor is still not identified, although mutagenesis experiments suggest that it should correspond to the E441 residue. In this work, we performed an extensive theoretical exploration of the potential energy surface of a model system representing the active site of RNR with FMCDP, using Density Functional Theory. This study establishes the detailed mechanism of inhibition, which is considerably different from the one proposed earlier. The proposed mechanism is fully consistent with available experimental data. Energetic results reveal unambiguously that the residue adding to the inhibitor is a cysteine, most probably C439, and exclude the possibility of the addition of E441. However, the E441 residue is shown to be essential for inhibition, catalyzing both the major and minor inhibition pathways, in agreement with mutagenesis results. It is shown also that the major mode of inactivation mimics the early stages of the natural substrate pathway.
Fernandes
Pedro Alexandrino
PA
CEQUP/ Faculdade de Ciências do Porto, Rua do Campo Alegre, 687, Portugal.
Ramos
Maria João
MJ
D001665:Binding Sites; D003845:Deoxycytosine Nucleotides / Q000737:chemistry* / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D008958:Models, Molecular; D008968:Molecular Conformation; D011487:Protein Conformation; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013816:Thermodynamics
D003845:Deoxycytosine Nucleotides; D004791:Enzyme Inhibitors; C100372:2'-fluoromethylene-2'-deoxycytidine 5'-diphosphate; D012264:Ribonucleotide Reductases