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Initial steady-state velocities using highly purified deuterated substrates and coenzymes as well as an isoenzymatically homogeneous enzyme preparation were measured for liver alcohol dehydrogenase at 25 deg in 20 mm phosphate (pH 7.0) or 20 mm pyrophosphate (pH 8.8). Significant isotope effects of v/sub 0,H//v/sub 0,D/ were observed. In the reduction of acetaldehyde by A- NADD the isotope effects were found to decrease with increasing acetaldehyde concentration, a result consistent with an ordered Theorell-- Chance mechanism for the reduction of acetaldehyde only at saturating substrate concentrations. No kinetically significant interactions were observed between the enzyme and the untransferred hydrogen of NADH. (auth) more...

Determination of dissociation constants by two different methods yield the following mean values in 20 millimolar phosphate, pH 7.0, 25 degrees C: 0.27 micromolar for reduced nicotinamide adenine dinucleotide (NADH); 0.29 micromolar for NADH with deuterium in the nicotinamide 4-B position (B-NADD); and 0.46 micromolar for NADH with deuterium in the nicotinamide 4-A position (A-NADD). These results indicate that dehydrogenases are capable of recognizing and distinguishing the appropriate hydrogen in the coenzyme already in the initial binding reaction. more...