Constitutive Inorganic Pyrophosphatase of Escherichia coli II. NATURE AND BINDING OF ACTIVE SUBSTRATE AND THE ROLE OF MAGNESIUM

摘要: Abstract 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 which the concentrations free pyrophosphate, Mg2+pyrophosphate, (Mg2+)2pyrophosphate estimated from determined association constants. The resultant data fit well a model was competitive inhibitor (Ki = 10-7 m), Mg2+pyrophosphate substrate (Km 5 x 10-6 bound (as inhibitor) relatively weakly if at all. Binding tri- tetrapolyphosphates, also substrates this enzyme, studies inhibition; their approximate binding constants 10-5 10-4 m, respectively. During enzymatic hydrolysis these polyphosphates, no intermediates could be detected. It is concluded that active site carries one or more positive charges it limited dimensions. structure substrate, proposed wherein divalent cation closes stable, 6-membered ring complex restricts rotation about phosphoanhydride bonds. If catalytic action entails motion fixation conceivably facilitate substrate.