ESCHERICHIA-COLI ORF17 CODES FOR A NUCLEOSIDE TRIPHOSPHATE PYROPHOSPHOHYDROLASE MEMBER OF THE MUTT FAMILY OF PROTEINS - CLONING, PURIFICATION, AND CHARACTERIZATION OF THE ENZYME

The product of the Escherichia coli orf17 gene is a novel nucleoside t riphosphate pyrophosphohydrolase with a preference for dATP over the o ther canonical (deoxy)nucleoside triphosphates, and it catalyzes the h ydrolysis of dATP through a nucleophilic attack at the beta-phosphorus to produce dAMP and inorganic pyrophosphate. It has a pH optimum betw een 8.5 and 9.0, a divalent metal ion requirement with optimal activit y at 5 mm Mg2+, a K-m of 0.8 mm and a k(cat) of 5.2 s(-1) at 37 degree s C for dATP. dAMP is a weak competitive inhibitor with a K-i of appro ximately 4 mw, while PPi is a much stronger inhibitor with an apparent K-i of approximately 20 mu M. The enzyme contains the highly conserve d signature sequence GXVEX(2)ETX(6)REVXEEX(2)I designating the MutT fa mily of proteins. However, unlike the other nucleoside triphosphate py rophosphohydrolases with this conserved sequence, the Orf17 protein do es not complement the mutT(-) mutator phenotype, and thus must serve a different biological role in the cell.