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JBC, Vol. 250, Issue 6, 1959-1965, Mar, 1975

Coenzyme interaction with horse liver alcohol dehydrogenase. Evidence for allosteric coenzyme binding sites from thermodynamic equilibrium studies

I. Iweibo and H. Weiner

The techniques of fluorescence enhancement, fluorescence quenching, fluorescence polarization, and equilibrium dialysis are utilized to study the binding properties of coenzyme to horse liver alcohol dehydrogenase. Polarization of fluorescence and equilibrium dialysis show that NADH binds to alcohol dehydrogenase with a stoichiometry of 6 mol per mol of enzyme, in contrast to the value of 2 determined from fluorescence enhancement measurements. NAD+ also binds with a stoichiometry of six as was determined by equilibrium dialysis. The two NADH sites which bind coenzyme more tightly and which are revealed by fluorescence enhancement measurements are designated the catalytic sites. Binding of coenzyme to the four ancillary sites does not alter the quantum yield of NADH but results in a 20% contribution to quenching of enzyme's tryptophan fluorescence. From the emission anisotropy of bound NADH of 24.0% for the additional sites and 28.1% for the catalytic sites and their relative fluorescence lifetimes at the same wavelengths of excitation and emmision, we conclude that the nicotinamide ring of NADH bound to the additional sites exhibits a freedom of motion independent of the macromolecule, while that bound to the catalytic sites is more rigidly held. Polarization of fluorescence yields negative intrinsic free energies of 9.2 and 7.5 Cal M-1 for NADH interaction with the catalytic and additional sites, respectively. Although these values are 1.3 to 2.0 Cal higher than those determined by fluorescence quenching and equilibrium dialysis, the mean Hill coefficient of 1.76 plus or minus 0.06, the titration span of 2.4 logarithmic units and coupling free energies (in magnitude and sign) are the same for all these techniques. The above difference in the intrinsic free energies are attributed largely to the different modes of interaction of excited and unexcited NADH molecules with alcohol dehydrogenase.
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