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

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Abstract
Beef heart lactate dehydrogenase has anion binding sites with selective affinities for F- and Cl-. F- competitively inhibits the catalytic activity of the enzyme and appears in a ternary enzyme-NADH-F- complex detectable in fluorescence and circular dichroism measurements. The presence of F- augments NADH binding, with a free energy of stabilization of -0.8 kcal/mol. NADH and Cl- are strongly antagonistic, in fact, almost mutually exclusive or competitive, in their interaction with beef heart lactate dehydrogenase. 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 Cl- 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 Cl- on the fluorescence and circular dichroism spectra of the bound NADH suggest that it binds primarily at sites other than the one occupied by F-.