Lactate Dehydrogenase of Lobster (Homarus americanus) Tail Muscle: II. Kinetics and regulatory properties

Abstract

Abstract Kinetic studies on the purified lobster muscle lactate dehydrogenase indicate that the reaction catalyzing the oxidation of lactate by DPN+ is modulated by the product DPNH. At low concentrations DPNH activates the reaction and at higher concentrations inhibits it. The combined effects of positive and negative modulations in activity result in sigmoidal rate patterns and rate plotted against DPN+ or l-lactate concentrations yields sigmoidal plots. The sigmoid patterns are converted to rectangular hyperbolas when the reaction is carried out in the presence of added reduced diphosphopyridine nucleotide. The kinetic features of the reaction catalyzing the oxidation of lactate by DPN+ have the general characteristics of second order autocatalyzed reactions. The reduced diphosphopyridine nucleotides are specific modulators of the reaction, the 3-acetylpyridine analogue of DPNH being a more effective modulator. The reaction catalyzing the reduction of pyruvate by DPNH follows normal Michaelis-Menton kinetics. A model is proposed based on the concept of subunit interactions controlling enzymatic activity to explain DPNH-effected modulations in the rate of lactate oxidation by DPN+. The oxidized and reduced forms of the diphosphopyridine nucleotides occupy the same sites on the protein; hence, in the proposed model a separate and distinct regulatory site for the effector molecule is not implicated. It is proposed that the binding of the reduced nucleotide to one or more of the four binding sites on the enzyme causes a structural change in the protein so that the binding of the oxidized nucleotide to another available site is facilitated. Such a model provides a reasonable explanation to account for the anomalous kinetics of the lobster lactate dehydrogenase. Product-induced positive and negative modulations in the rate of lactate oxidation provide the system with a built-in regulatory mechanism whereby the product DPNH controls the rate of its own production.