CHARACTERIZATION OF NUCLEOTIDE DEPENDANT OLIOGOMERIC STATES OF RNR1P USING SAXS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in production of the pool of deoxyribonucleotides necessary for DNA replication. Crucial for rapidly proliferating cells, RNR is a successful target for anti-HSV and anticancer drugs. Allosteric regulation consitutes one of the important modes of regualtion of RNR activity.Currently, there are two major models that describe the allosteric behavior of RNR. The model proposed by Reichard and Thelander (RT model) accounts that the substrate binding at the catalytic site (c-site) is governed by a given dNTP bound to the specificity site (s-site), while global enzyme activity is dependent on either ATP or dATP binding to the activity site(Eriksson, Uhlin et al. 1997). One of the major drawbacks of the RT model is its inability of explaining RNR's activity in the context of subunit olgiomerization. A recently described comprehensive allosteric model by Cooperman not only takes in the basic tenets of the RT model but also qualitatively describes how enzyme activity is regulated by nucleotide dependant oligomerization. In addition to the s- and a-sites, the comprehensive model describes a third allosteric site in Rnr1 known as the hexameric site (h-site). At physiologically significant concentrations, binding of ATP to h-site drives hexamer formation. In this model, dATP is regarded as a universal inhibitor because of inactive tetramer formation. In this proposal we hope to characterize the nucleotide dependant oligomerization state of Rnr1 subunit of the RNR complex using small angle X-ray scattering.