KINETIC CHARACTERISTICS OF NUCLEOSIDE MONO-PHOSPHATASE, DI-PHOSPHATASE AND TRIPHOSPHATASE ACTIVITIES OF THE PERIPLASMIC 5'-NUCLEOTIDASE OF ESCHERICHIA-COLI

Periplasmic 5'-nucleotidase from Escherichia cell, in addition to the monophosphoesterase activity has a diphosphohydrolase activity, acting on nucleoside di- and triphosphates. We proposed that the monophospho esterase and diphosphohydrolase activities have their own active site. This proposal is based on the different types of bonds being broken. Chemical modification with selective group reagents did not show diffe rences in the essentiality of some residues, like histidyl, carboxyl a nd arginyl groups, of these two hydrolytic activities. While kinetic a pproaches employing the competition plot and unidirectional substrate inhibition point to that diphosphohydrolase activity (ATPase-ADPase) d o not share the same active site with monophosphoesterase activity. We stern blotting developed with polyclonal anti-placental apyrase antibo dy revealed a single protein in the periplasmic fraction of 66.5 kDa s imilar to the Mr of the purified enzyme by isoelectrofocusing. (C) 199 7 Elsevier Science Inc.