Ribonucleotide reductase initiates de nova DNA biosynthesis through the reduction of nucleotides to their corresponding 2$\sp\prime$-deoxynucleotides. The active reductase is composed of a catalytic portion (the R1 enzyme) and a cofactor (R2). All ribonucleotide reductases accept four nucleotide substrates. Activity toward a given substrate is dictated by the binding of nucleotide allosteric effectors. This dissertation describes the first comprehensive quantitative model for the regulation of ribonucleotide reductase from any source. The model incorporates information about R2 protein cofactor, nucleotide substrate and allosteric effector binding, enzyme quaternary structure and activity to provide a mechanism for the effector dependent modulation of the activity of ribonucleotide reductase.
The proposed model consists of twenty one states for each substrate, where a state is defined by the quaternary structure of the R1 enzyme, the presence or absence of bound cofactor (R2) and substrate, and the number, location and identity of bound allosteric effectors. The results confirm that dimerization of R1 is essential for enzymatic activity. It is also shown that R1 dimerization is not sufficient for the reduction of substrate. Purine reduction by murine ribonucleotide reductase is shown to be regulated by modulation of the catalytic rate constant (allosteric V system). No heterotropic cooperativity between substrates and allosteric effectors is observed. Limited studies on the regulation of pyrimidine reduction support the same model.
The allosteric effectors ATP and dATP are shown to mediate the formation of the R1 tetramer. In the presence of the cofactor, ATP induced tetramer formation is a slow process. Tetramerization by either ATP or dATP leads to the formation of an enzymatically inactive species. However, the specific activity of pyrimidine reductases can be restored at very high ATP concentrations. Re-activation occurs in an ATP concentration range in which the R1 enzyme forms hexamers in dynamic light scattering experiments. Taken together, the data argue for the existence of an additional site for ATP, and, perhaps, an active form of the enzyme other than the dimer.
