Porcine Heart Lactate Dehydrogenase

The kinetics of NADH, 3-thionicotinamide adenine dinucleotide (TNAD), and oxamate binding to the H4 isoenzyme of lactate dehydrogenase from the pig has been investigated by temperature jump techniques. The dissociation rate constant for TNAD is considerably larger than for NADH, whereas the recombination rate constant is smaller for the oxidized coenzyme than for the reduced molecule. The kinetics of oxamate binding agrees satisfactorily with a simple binding mechanism. The optical rotatory dispersion spectrum of the protein, which indicates α-helical content, is unperturbed by binding of NADH and oxamate, or by changing the pH from 6 to 8. Both the enthalpy and entropy of oxamate binding are strongly pHdependent. The interaction of this inhibitor with the protein cannot be explained simply in terms of an electrostatic attraction. Other effects, primarily entropic, are of large importance. Evidence is presented which indicates that TNAD in solution exists in two conformational forms which are in rapid equilibrium.