Thermodynamic studies of the activation of rabbit muscle lactate dehydrogenase by phosphate.
In an attempt to trace the source of phosphate activation of the enzyme-catalysed pyruvate-lactate interconversion by rabbit muscle lactate dehydrogenase, equilibrium constants were measured to examine the effects of phosphate on interactions pertinent to the enzymic process. Frontal gel-chromatographic studies of the binding of NADH to the enzyme established that the intrinsic association constant is doubled in the presence of 50 mM-phosphate in the buffer (pH 7.4, I0.15). From kinetic studies of the competition between NAD+ and NADH for the coenzyme-binding sites of the enzyme it is concluded that the binding of oxidized nicotinamide nucleotide is also doubled in the presence of 50 mM-phosphate. Competitive-inhibition studies and fluorescence-quenching measurements indicated the lack of a phosphate effect on ternary-complex formation between enzyme-NADH complex and oxamate, a substrate analogue of pyruvate. The equilibrium constant for the interaction between enzyme-NAD+ complex and oxalate, an analogue of lactate, was also shown, by difference spectroscopy, to be insensitive to phosphate concentration. Provided that the effects observed with the substrate analogues mimic those operative in the kinetic situation, the equilibrium constant governing the isomerization of ternary complex is also independent of phosphate concentration. It is concluded that enhanced coenzyme binding is the source of phosphate activation of the rabbit muscle lactate dehydrogenase system.
D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D046911:Macromolecular Substances; D009132:Muscles / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D010070:Oxalates / Q000378:metabolism; D019815:Oxalic Acid; D010072:Oxamic Acid / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology*; D011817:Rabbits; D013816:Thermodynamics
D046911:Macromolecular Substances; D010070:Oxalates; D010710:Phosphates; D009243:NAD; D019815:Oxalic Acid; D007770:L-Lactate Dehydrogenase; D010072:Oxamic Acid