Investigations of the inhibition mechanisms of human ribonucleotide reductase by gemcitabine-5'-diphosphate and saccharomyces cerevisiae ribonucleotide reductase by Sml1

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2009.Vita. Cataloged from PDF version of thesis.Includes bibliographical references.Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides supplying the dNTPs required for DNA replication and DNA repair. Class I RNRs require two subunits ([alpha] and [beta]) for activity. The [alpha] subunit binds the substrates and the allosteric effectors that govern specificity and turnover. The 32 subunit houses the diferric Y* cofactor required to initiate nucleotide reduction. Human cells possess two type of P subunits of RNR: one ([beta]) is involved in DNA replication and the second (p53[beta]') is required for mitochondrial DNA replication and likely plays some role in DNA repair. Gemcitabine (2',2'-difluoro-2'-deoxycytidine, F2C) is used clinically in a variety of cancer treatments and the phosphorylated F2C targets many enzymes involved in nucleotide metabolism, including RNR. The studies presented here with [1 '-3H]- and [5- 3H]-F 2CDP have established that F2CDP is a sub-stoichiometric mechanism based inhibitor (0.5 equivalents F2CDP/[alpha]) of both the E. coli and the human RNRs in the presence of a reductant. Inactivation is caused by covalent labeling of RNR by the sugar of F2CDP (0.5 equivalents/[alpha]) and is accompanied by the release of 0.5 equivalent cytosine/[alpha]. Studies using size exclusion chromatography reveal that in the E. coli RNR, an u212 tight complex is generated subsequent to enzyme inactivation by F2CDP, while in the human RNR, an [alpha]6[beta]6 or [alpha]6[beta]'6 tight complex is generated. The second part of this thesis focuses on the Sml inhibition mechanism in S. cerevisiae. Smll is a 12 kDa small protein RNR inhibitor.(cont.) It regulates RNR activity by binding directly to a to repress RNR activity. The binding of Smll to a has been proposed to block the reduction of the active site disulfide formed concomitantly with dNTP production, leaving a in the oxidized form. A fluorescence titration method was employed to measure the Kd of Smll with different forms of c. Our data suggest that Smll binds to a by a mechanism that involves its C-terminal helix (likely the hydrophobic face) and a region of a that includes W688. The kinetics studies suggest that Smll behaves as an uncompetitive inhibitor relative to the substrate, and binds to the oxidized form of [alpha] in preference to the reduced form.by Jun Wang.Ph.D