A kinetic study has been made of the reaction catalyzed by a purified preparation of nucleoside diphosphatase which has been subjected to prolonged storage at –10°. Such treatment has been shown to affect some, but not all, of the properties of the enzyme. Thus both preparations have approximately the same molecular weight and are activated by the allosteric modifier, magnesium adenosine triphosphate (MgATP2–). However, whereas the freshly prepared enzyme exhibits nonlinear double-reciprocal plots of velocity as a function of the magnesium inosine diphosphate (MgIDP–) concentration and its maximum velocity is reduced in the presence of the modifier, the aged enzyme gives linear plots of 1/v vs. 1/MgIDP– and its maximum velocity is unaffected by the addition of modifier. The kinetic data are consistent with a reaction mechanism in which it is proposed that the aged enzyme possesses distinct substrate and modifier sites, that there is random addition of one molecule (or independent combinations of multiple molecules) of each of these reactants to the enzyme under rapid equilibrium conditions and that the modifier facilitates the combination of substrate without influencing the rate of product formation. Values for the various kinetic constants have been determined.
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