Ribonucleotide reductases are uniquely responsible for the reduction of necleotides to deoxynucleotides and have been divided into two classes defined by the nature of their cofactors. One class is represented by the E. coli enzyme (RDPR-composed of B1 and B2 subunits) which requires 2 Fe+3 and an organic tyrosine radical for activity. The second class is represented by the L. leichmannii enzyme (RTPR composed of one subunit) and requires 5 feet-deoxyadenosyl cobalamin for activity. This proposal is concerned with a detailed study of the intriguing mechanisms of both classes of reductases and the use of this mechanistic information to design suicide inhibitors of these enzymes. Our working hypothesis has been that even though the cofactors of these two classes of enzymes are structurally different, that the chemical mechanisms of reduction are similar. Specifically, we have put forth a new mechanism for this reduction involving a radical cation intermedaite. The basic objectives of this proposal are: (1) to obtain evidence for or against a homolytic mechanism using rapid quench EPR spactroscopy, rapid scan spectroscopy and spin trapping experiments; (2) to study the nature of the active sties of both RDPR and RTPR using two unique classes of suicide inhibitors represented by 2 feet-chloro-2 feet-deoxyuridine 5 feet-di(tri)phosphate (ClUDP) and 2 feet-azido-2 feet-deoxy-uridine 5 feet-di(tri)phosphate. In the case of [5 feet-3H]ClUDP to isolate the peptide of RDPR and RTPR which is radiolabeled, to detemine the amino acid residue which has been modified and the structure of the sugar moiety attached to this residue; and (3) to design several unique classes of suicide inhibitors based on this mechanistic information: B1 inactivators, analogous to 2 feet-ClUDP; B2 inactivators, scavengers of the protein radical and B2 inactivators, scavengers of the substrate or product radicals. Based on the strong similarities between the E. coli and mammalian reductases, these inhibitors may function as antitumor, antiviral, or antiparasitic agents.
Showing the most recent 10 out of 125 publications
