Use of the sulphite adduct of nicotinamide–adenine dinucleotide to study ionizations and the kinetics of lactate dehydrogenase and malate dehydrogenase

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Abstract
The formation of the non-enzymic adduct of NAD+ and sulfite was investigated. In agreement with others it is concluded that the dianion of sulfite adds to NAD+. The formation of ternary complexes of either [pig heart] lactate dehydrogenase [EC 1.1.1.27] or malate dehydrogenase [EC 1.1.1.37] with NAD+ and sulfite was investigated. The UV spectrum of the NAD-sulfite adduct was the same whether free or enzyme-bound at either pH 6 or pH 8. This suggests that the free and enzyme-bound adducts have a similar electronic structure. The effect of pH on the concentration of NAD-sulfite bound to both enzymes was measured in a new titration apparatus. Unlike the non-enzymic adduct (where the stability change with pH simply reflects HSO3- = SO32- + H+), the enzyme-bound adduct showed a bell-shaped pH-stability curve, which indicated that an enzyme side chain of pK = 6.2 must be protonated for the complex to form. Since the adduct does not bind to the enzyme when histidine-195 of lactate dehydrogenase is ethoxycarbonylated it is concluded that the protein group involved is histidine-195. The pH-dependence of the formation of a ternary complex of lactate dehydrogenase, NAD+ and oxalate suggested that an enzyme group is protonated when this complex forms. The rate at which NAD+ binds to lactate dehydrogenase and malate dehydrogenase was measured by trapping the enzyme-bound NAD+ by rapid reaction with sulfite. The rate of NAD+ dissociation from the enzymes was calculated from the bimolecular association kinetic constant and from the equilibrium binding constant and was in both cases much faster than the forward Vmax. No kinetic evidence was found that suggested that there were interactions between protein subunits on binding NAD+.