Human lactate dehydrogenase A undergoes allosteric transitions under pH conditions inducing the dissociation of the tetrameric enzyme

The aerobic energetic metabolism of eukaryotic cells relies on the glycolytic generation of pyruvate, which is subsequently channelled to the oxidative phosphorylation taking place in mitochondria. However, under conditions limiting oxidative phosphorylation, pyruvate is coupled to alternative energetic pathways, e.g. its reduction to lactate catalyzed by lactate dehydrogenases (LDHs). This biochemical process is known to induce a significant decrease in cytosolic pH, and is accordingly denoted lactic acidosis. Nevertheless, the mutual dependence of LDHs action and lactic acidosis is far from being fully understood. Using human LDH-A, here we show that when exposed to acidic pH this enzyme is subjected to homotropic allosteric transitions triggered by pyruvate. Conversely, human LDH-A features Michaelis-Menten kinetics at pH values equal to 7.0 or higher. Further, citrate, isocitrate, and malate were observed to activate human LDH-A, both at pH 5.0 and 6.5, with citrate and isocitrate being responsible for major effects. Dynamic light scattering (DLS) experiments revealed that the occurrence of allosteric kinetics in human LDH-A is mirrored by a consistent dissociation of the enzyme tetramer, suggesting that pyruvate promotes tetramer association under acidic conditions. Finally, using the human liver cancer cell line HepG2 we isolated cells featuring cytosolic pH equal to 7.3 or 6.5, and we observed a concomitant decrease in cytosolic pH and lactate secretion. Overall, our observations indicate the occurrence of a negative feedback between lactic acidosis and human LDH-A activity, and a complex regulation of this feedback by pyruvate and by some intermediates of the Krebs cycle.

Automated summary

The study reveals that human LDH-A undergoes homotropic allosteric transitions when exposed to acidic pH, while displaying Michaelis-Menten kinetics at pH values equal to or higher than 7.0. Additionally, citrate, isocitrate, and malate activate human LDH-A, with citrate and isocitrate leading to major effects, and the allosteric kinetics in human LDH-A is accompanied by the dissociation of the enzyme tetramer under acidic conditions. Furthermore, a negative feedback between lactic acidosis and human LDH-A activity is observed, indicating a complex regulation of this feedback by pyruvate and intermediates of the Krebs cycle.