Use of the sulphite adduct of nicotinamide-adenine dinucleotide to study ionizations and the kinetics of lactate dehydrogenase and malate dehydrogenase.
1. The formation of the non-enzymic adduct of NAD(+) and sulphite was investigated. In agreement with others we conclude that the dianion of sulphite adds to NAD(+). 2. The formation of ternary complexes of either lactate dehydrogenase or malate dehydrogenase with NAD(+) and sulphite was investigated. The u.v. spectrum of the NAD-sulphite adduct was the same whether free or enzyme-bound at either pH6 or pH8. This suggests that the free and enzyme-bound adducts have a similar electronic structure. 3. The effect of pH on the concentration of NAD-sulphite bound to both enzymes was measured in a new titration apparatus. Unlike the non-enzymic adduct (where the stability change with pH simply reflects HSO(3) (-)=SO(3) (2-)+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 we conclude that the protein group involved is histidine-195. 4. 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. 5. The rate at which NAD(+) binds to lactate dehydrogenase and malate dehydrogenase was measured by trapping the enzyme-bound NAD(+) by rapid reaction with sulphite. 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 V(max.). No kinetic evidence was found that suggested that there were interactions between protein subunits on binding NAD(+).
D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase; D008291:Malate Dehydrogenase; D009243:NAD / Q000031:analogs & derivatives*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013447:Sulfites
D013447:Sulfites; D009243:NAD; D006639:Histidine; D007770:L-Lactate Dehydrogenase; D008291:Malate Dehydrogenase