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

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.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2009

Author dc:creator

• Wang, Jun, Ph. D. Massachusetts Institute of Technology

Advisor dc:contributor.advisor

• JoAnne Stubbe.

• Chemistry.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/55099

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/55099