A highly purified preparation of nucleoside diphosphatase has been used to study the kinetics of the reaction at pH 8.5 under conditions where magnesium-inosine diphosphate was the variable substrate and the concentration of inosine diphosphate was controlled. Double-reciprocal plots of initial velocity as a function of the magnesium-inosine diphosphate concentration at a relatively low, fixed concentration of inosine diphosphate yielded concave-up nonrectangular hyperbolas. When present at a sufficiently high concentration, the allosteric activator, magnesium-adenosine triphosphate, caused this type of plot to become linear. The reaction velocities at lower substrate concentrations were increased by the addition of magnesium-adenosine triphosphate while those at relatively high substrate concentrations were decreased. Studies of the activation showed that the velocity increased as a hyperbolic function of the modifier concentration. The results are in agreement with, and have been interpreted according to, a rapid equilibrium, random mechanism that allows for the interdependent reaction of two molecules of substrate, at two identical catalytic sites on the enzyme, to yield complexes that give rise to products at different rates. The mechanism also assumes that magnesium-adenosine triphosphate combines at a single, distinct site on the enzyme, so that it can react with free enzyme and all the enzyme-substrate complexes. It is further proposed that the modifier can influence the binding of substrate as well as the rate of product formation. Mathematical analysis of the data has shown that the experimental results are consistent with the suggested mechanism and values have been determined for the various kinetic constants. The results have also been discussed in relation to other theories for the mechanism of allosteric enzyme action.
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