The V of horse liver aldehyde dehydrogenase is enhanced twofold in the presence of 0.5 m m Mg 2+ ions when assayed in the dehydrogenase reaction. The mechanism of this activation appears to be related to the fact the enzyme changes from functioning with half-of-the-sites reactivity to functioning with all-of-the-sites reactivity. That is, the presteady-state burst magnitude increases from 2 mol NADH formed per mole of tetrameric enzyme to 4 mol formed per mole ( K. Takahashi and H. Weiner, J. Biol. Chem. , 1980 , 255 , 8206–8209). Whether this twofold enhancement correlates, in fact, to a change from half-of-the-sites to all-of-the-sites reactivity of the enzyme by Mg 2+ ions was investigated by determining the Stoichiometry of coenzyme binding by fluorescence quenching and enhancement methods in the absence and presence of the metal ions. The biphasic Scatchard plots for NAD binding to the enzyme were similar in the absence and presence of Mg 2+ ions, while that of NADH binding was monophasic (-Mg 2+ ) and biphasic (+Mg 2+ ). In the presence of p -methoxyacetophenone, a competitive inhibitor for substrate, the stoichiometric titration of coenzyme binding to the ternary complexes (enzyme-NAD(H)-inhibitor) revealed that only 2 mol of NAD or NADH bind in the absence of Mg 2+ ions but 4 bind per mole of tetrameric enzyme in the presence of added metal. The fluorescence intensity of NAD's fluorescent derivative, 1, N 6 -ethenoadenine dinucleotide, bound to the enzyme was also doubled by the addition of Mg 2+ ions. The combined binding data show that the stoichiometry of coenzyme binding to aldehyde dehydrogenase in the ternary complex increases from 2 to 4 mol binding per mole of tetrameric enzyme with the addition of Mg 2+ ions. This increase in stoichiometry corresponds to the observed changes of burst magnitude obtained from the presteady-state and V in the steady-state kinetics assays. From both results of the kinetics and stoichiometry, we show that horse liver aldehyde dehydrogenase exhibits half-of-the-sites reactivity when in the tetrameric state in the absence of Mg 2+ ions, and all-of-the-sites reactivity in the dimeric state in the presence of the metal.
