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

Abstract

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.