Constitutive Inorganic Pyrophosphatase of Escherichia coli

Abstract

Abstract An intracellular, inorganic pyrophosphatase from Escherichia coli K-12 3000(λ) has been purified 500-fold to a state of apparent homogeneity. The enzyme catalyzes hydrolysis of inorganic pyrophosphate, tripolyphosphate, and tetrapolyphosphate with relative velocities of 1.000, 0.016, and 0.007, respectively. No activity whatsoever was found with a variety of other phosphate esters. There was an absolute requirement for divalent cation in amounts suggesting stoichiometric combination with the substrate. At pH 9.1, Mg2+ and Mn2+ were maximally effective; at pH 7.5, Zn2+ and Co2+ were best. Although no exchange reaction (32Pi ⇄ PPi) could be demonstrated, a net reversal of the reaction was achieved by coupling through thymidine diphosphate glucose pyrophosphorylase and phosphoglucomutase to oxidation of glucose 6-phosphate to 6-phosphogluconic acid. The stoichiometry of the reverse reaction indicated utilization of 10% of the 32Pi (trapped as thymidine triphosphate). The enzyme was exceptionally stable. In the presence of 0.01 m Mg2+, it withstood a temperature of 80° for 10 min.