Effects of halides on reduced nicotinamide adenine dinucleotide binding properties and catalytic activity of beef heart lactate dehydrogenase.

Beef heart lactate dehydrogenase has anion binding sites with selective affinities for fluoride and chloride. Fluoride competitively inhibits the catalytic activity of the enzyme and appears in a ternary enzyme-reduced nicotinamide adenine dinucleotide (NADH)-fluoride complex detectable in fluorescence and circular dichroism measurements. The presence of fluoride augments NADH binding, with a free energy of stabilization of -0.8 kcal/mol. NADH and chloride are strongly antagonistic, in fact, almost mutually exclusive or competitive, in their interaction with beef heart lactate dehydrogenase. In addition, the Hill coefficient for NADH binding undergoes a small but repeatable decline, reaching a minimum value of 0.75-0.8 at physiological NaCl concentrations. Dilution experiments showed that NADH binding in the presence of NaCl is independent of enzyme concentration, demonstrating that the chloride sensitivity is not linked to reversible dissociation of the enzyme. The NADH binding equilibria determined in NaCl, KCl, or CsCl are identical. The minimal effects of chloride on the fluorescence and circular dichroism spectra of the bound NADH suggest that it binds primarily at sites other than the one occupied by fluoride.