Site-directed Mutagenesis and HPLC Analysis of Inosine-Uridine Nucleoside Hydrolase RihC of <italic>Escherichia coli</italic>

Abstract

Recycling of purine and pyrimidine bases is critical in the metabolism of many organisms. While the archetype enzyme, Inosine-Uridine Nucleoside Hydrolase (IU-NH) of <italic>Crithidia fasciculata</italic>, is well studied because of its potential as a therapeutic target, other enzymes have not been well characterized, including ribonucleoside hydrolase C (RihC) of <italic>Escherichia coli</italic>. This study examined the effects of amino acid changes at conserved residues of RihC that have previously been shown to be critical for functioning of a similar enzyme in the protozoan <italic>C. fasciculata</italic>. Mutagenized RihC proteins, purified to greater than 95% purity, showed significant differences in kinetics when assayed by HPLC for uridine and inosine hydrolysis. Changes at four amino acid residues resulted in reductions in velocity when compared to the wild type enzyme for hydrolysis of uridine. These results support the critical roles for these residues for enzyme activity.