KINETIC MECHANISM OF NUCLEOSIDE HYDROLASE FROM ESCHERICHIA. COLI

Abstract

Nucleoside hydrolases are key enzymes that catalyze the hydrolysis of the N-glycosidic bond in nucleosides to allow recycling of the purines and pyrimidines.1 Inosine-Uridine Nucleoside Hydrolase (IU-NH) of Crithidia fasciculata has previously been studied and considered as a potential target for drug design. Although various nucleoside hydrolases have been extensively characterized many aspects of the ribonucleoside hydrolase C (RihC), are unclear. In this study, wild type and mutants of RihC from E.coli have been expressed, purified, and analyzed. Gel electrophoresis confirmed the purity and the existance of the proteins as tetramers. The calculated molecular weight based on the amino acid sequence is 32.57 kDa while SDS-PAGE indicated a molecular weight of 33 kDa. Analyzed proteins showed significant activity with the substrate inosine. Turnover number and specific activity were consistent with each other. A 3D structure of RihC from E.coli bound with substrate inosine was developed using CLC Drug Discovery workbench software. Interactions between the substrate and residues involved in the active site of the enzyme were identified. A comparison between the known structure of IU-NH from C. fasciculata and the modeled RihC from E.coli with inosine bound in the active sites was carried out with regard to the interactions between the substrate and the residues in the active site.