Purification, characterization, and activity of chimeric Escherichia coli/mouse and Plasmodium falciparum small subunits of Type I ribonucleotide reductase

Ribonucleotide reductase catalyzes the conversion of ribonucleotides to deoxyribonucleotides, reducing the 2$\sp\prime$-hydroxyl group to hydrogen. This enzyme is the only de novo source of deoxynucleotide precursors required for deoxyribonucleic acid synthesis. Type I reductases, found in Escherichia coli, eucaryotes, and vaccinia virus, are composed of two nonidentical dimeric subunits, designated large and small based on their relative sizes. The large subunit contains the substrate and effector binding sites. The small subunit contains a required $\mu$-oxo-bridged dinuclear iron center and stable tyrosyl free radical. Subunit association is required for enzymatic activity. The large subunit binding site has been localized to the carboxy-terminus of the small subunit. These carboxy-terminal sequences are species-specific, and there is no cross-reactivity of large and small subunits from species with different sequences. For this thesis, two chimeric proteins were designed to test whether the presence of both required portions of a Type I small subunit were sufficient for catalytic activity. The two chimeric proteins were prepared, containing the dinuclear iron/tyrosyl radical center portion of R2 from Escherichia coli and the carboxy-terminal binding sequence from mouse. Two mouse carboxy-terminal sequences were used, seven or thirty-three amino acids in length. Both proteins were overexpressed and purifed from an Escherichia coli system. The chimeras had nearly identical physical characteristics to the Escherichia coli small subunit, as determined by circular dichroism, ultraviolet-visible, and electron paramagnetic resonance spectroscopies. However, the chimeras were found not to be catalytically active when assayed with the mouse large subunit. Instead, the chimeric small subunits were found to bind competitively the large subunit of mouse ribonucleotide reductase in a 1:1 subunit stoichiometry. Possible reasons for lack of activity, including distorted binding and loss of intersubunit long-range electron transfer, are discussed. In addition, the known mammalian ribonucleotide reductase inhibitor, a seven amino acid peptide with the sequence of the small subunit carboxy-terminus, was shown to bind the large subunit in a 1:2 large subunit:peptide stoichiometry. Furthermore, the expression, purification, and activity of the small subunit of the Type I ribonucleotide reductase from Plasmodium falciparum, the deadliest human malarial parasite, are presented. The studies presented herein demonstrate that the malarial small subunit is active when assayed with the mouse large subunit. This activity is inhibited by the same peptide that inhibits mouse ribonucleotide reductase, as described above. The large and small subunits from these different species bind one another and are catalytically active; this result is discussed in terms of the homology of their small subunit carboxy-terminal sequences. As an adjunct to work on the malarial small subunit, the expression and purification of the mouse small subunit were optimized. The results of these studies are presented