The first committed step in DNA biosynthesis occurs by direct reduction of
ribonucleotides. This reduction is catalyzed by ribonucleotide reductase (RNR), an
enzyme which uses a unique radical mechanism to facilitate the transformation. All four
DNA precursors are synthesized by a single enzyme. Therefore, an intricate pattern of
regulation has evolved to insure that RNR generates the proper quantity of each
deoxyribonucleotide. It is this regulation, and conditions that influence this regulation,
that are the central focal points of this dissertation.
The studies described in this thesis have been aided by the development of a novel
RNR assay. Unlike the traditional assay, this new procedure permits the simultaneous
monitoring of all four RNR activities. This four-substrate assay was used to investigate
whether the four enzyme activities of RNR were differentially sensitive to inhibition by the
radical scavenger, hydroxyurea. The assay results, along with the results of a technique
that measured enzyme inhibition as a function of radical decay, suggest that all activities of
RNR are equally inhibited by hydroxyurea. Instead of differential inhibition, it appears
that the activity level of RNR determines the relative sensitivity to hydroxyurea.
The effects of nucleotide effectors and substrates on the relative turnover rates of the
vaccinia virus and T4 phage RNR were also investigated by use of the four-substrate
assay. When physiological concentrations of the allosteric effectors and substrates were
added to the reaction mixtures, both enzyme forms produced dNDPs in ratios that
approximate the nucleotide composition of their respective genomes. Non-physiological
nucleotide concentrations generated significantly different product profiles, indicating that
RNR has evolved to function within a defined nucleotide environment. Interestingly, the
substrate component of the nucleotide environment proved to be as important as the
allosteric effectors in modulating the reaction rates. Although the allosteric effects of
nucleoside triphosphates have been known for some time, little attention has been given to
the potential role that substrates play in the regulation of RNR. The results from my
research suggest that the regulation of RNR in vivo results from a complex interplay
between the enzyme and its substrates, products, and allosteric effectors. / Graduation date: 1998
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/33884 |
| Date | 12 March 1998 |
| Creators | Hendricks, Stephen P. |
| Contributors | Mathews, Christopher K. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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