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Constitutive Inorganic Pyrophosphatase of Escherichia coli

II. NATURE AND BINDING OF ACTIVE SUBSTRATE AND THE ROLE OF MAGNESIUM

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

A plausible kinetic scheme for interaction of Escherichia coli inorganic pyrophosphatase with pyrophosphate and its magnesium complexes has been deduced by computer model-fitting procedures. Zero order enzyme velocities were measured in pH 9.1 solutions in which the concentrations of free pyrophosphate, Mg²⁺pyrophosphate, and (Mg²⁺)₂pyrophosphate were estimated from determined association constants. The resultant data fit well a model in which free pyrophosphate was competitive inhibitor (K_i = 10^-7 m), Mg²⁺pyrophosphate was substrate (K_m = 5 x 10^-6 m), and (Mg²⁺)₂pyrophosphate was bound (as a competitive inhibitor) relatively weakly if at all.

Binding of inorganic tri- and tetrapolyphosphates, also substrates for this enzyme, was estimated by studies of competitive inhibition; their approximate binding constants were 10^-5 and 10^-4 m, respectively. During enzymatic hydrolysis of these polyphosphates, no free intermediates could be detected.

It is concluded that the active site of the enzyme carries one or more positive charges at pH 9.1 and that it is of limited dimensions. A structure for Mg²⁺pyrophosphate, the active substrate, is proposed wherein the divalent cation closes a stable, 6-membered ring in the complex and restricts rotation about the pyrophosphate phosphoanhydride bonds. If catalytic action entails motion of the enzyme, this fixation could conceivably facilitate hydrolysis of bound substrate.

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