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Constitutive Inorganic Pyrophosphatase of Escherichia coli

I. PURIFICATION AND CATALYTIC PROPERTIES

From the ¹ From the Department of Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110

An intracellular, inorganic pyrophosphatase from Escherichia coli K-12 3000() 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 (³²P_i PP_i) 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 ³²P_i (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.

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