Effect of macromolecular crowding on the kinetics of enzymatic reactions catalysed by oligoproteins. The LDH dimer-tetramer case.

Abstract

A study on the enzyme kinetics of L-lactate dehydrogenase, which catalyses the reduction of pyruvate to lactate oxidizing NADH, is presented. This reaction occurs when a lack of oxygen is present and is related to muscular fatigue. LDH is one of the most important biomarkers of injuries and disease, because it is released during tissue breakdown. General concepts of enzyme kinetics have been reviewed and some models to explain the kinetics of the enzyme have been proposed. L-lactate dehydrogenase is a tetrameric protein, an enzyme formed by four subunits, and the presence of a possible cooperativity, i.e., different affinity in each active centre, must be considered. Besides, macromolecular crowding, the alteration of the behaviour of molecules with the presence of highly concentrated macromolecules, and its possible effects on enzyme kinetics have been presented. A series of experiments, measuring the initial velocity of the reaction by spectrophotometric means and using a stopped-flow methodology, have been performed. The experiments have been carried out varying the pyruvate and the enzyme concentration and working in solution conditions. A series of experiments in crowded media, at high macromolecules concentration, have been performed. The crowded media experiments have been carried out using different obstacle sizes, using dextran polymer to simulate the cellular crowding, and with different enzyme concentrations. A surface plot of initial velocity as a function of substrate and enzyme concentrations, for the solution media data, has been obtained. The data has been fitted to the proposed models and the results have suggested an ideal behaviour without cooperativity. In crowded media, a higher decrease in the reaction velocity has been found when using the bigger dextran and the higher enzyme concentration. An auto-crowding hypothesis, the enzyme acts itself as a crowding agent, is presented to possibly explain the results.