Purification and properties of cyclic phosphodiesterase: 3′-nucleotidase, a periplasmic enzyme of Haemophilus influenzae

Abstract

The cyclic phosphodiesterase: 3′-nucleotidase of Haemophilus influenzae catalyzes the cleavage of 2′,3′-cyclic nucleotides to 3′-monoesters, and the hydrolysis of the resulting 3′-nucleotides to nucleosides and inorganic phosphate. It also hydrolyzes the artificial substrate bis( p-nitrophenyl)phosphate, yielding p-nitrophenol and p-nitrophenyl phosphate. The enzyme is located in the periplasmic space of H. influenzae, and can be selectively released by subjecting the cells to osmotic shock, or by converting the cells to spheroplasts. The selective removal of the enzyme from H. influenzae was effected by incubating the cells in a solution of Tris, EDTA, and Nonidet P-40; the enzyme was further purified to apparent electrophoretic homogeneity. The enzyme from H. influenzae hydrolyses 3′-nucleotides in the absence of any added metal ion, although Co 2+ and Ca 2+ stimulate the cleavage of bis( p-nitropheny])phosphate. We have observed a marked competitive inhibition of bis( p-nitrophenyl)phosphate cleavage by 2′-, 3′-, and 5′-adenosine monophosphate. The 2′- and 5′-nucleotides also inhibit the cyclic phosphodiesterase and 3′-nucleotidase activities. These results indicate that nucleotide substrates and the artificial chromogenic substrate interact with the same active site.