Acetyl-Coenzyme A Can Regulate Activity of the Mitochondrial Pyruvate Dehydrogenase Complex in Situ

Abstract

In vitro, the pyruvate dehydrogenase complex is sensitive to product inhibition by NADH and acetyl-coenzyme A (CoA). Based upon Km and K1 relationships, it was suggested that NADH can play a primary role in control of pyruvate dehydrogenase complex activity in vivo (JA Miernyk, DD Randall [19871 Plant Physiol 83: 306-310). We have now extended the in vitro studies of product inhibition by assaying pyruvate dehydrogenase complex activity in situ, using purified intact mitochondria from green pea (Pisum sativum) seedlings. In situ activity of the pyruvate dehydrogenase complex is inhibited when mitochondria are incubated with malonate. In some instances, isolated mitochondria show an apparent lack of coupling during pyruvate oxidation. The inhibition by malonate, and the apparent lack of coupling, can both be explained by an accumulation of acetyl-CoA. Inhibition could be alleviated by addition of oxalacetate, high levels of malate, or Lcamitine. The CoA pool in nonrespiring mitochondria was approximately 150 micromolar, but doubled during pyruvate oxidation, when 60 to 95% of the total was in the form of acetyl-CoA. Our results indicate that in situ activity of the mitochondrial pyruvate dehydrogenase complex can be controlled in part by acetyl-CoA product inhibition.The mPDC' occupies a crucial branch point in intermediary metabolism, and activity is regulated by several mechanisms including compartmentation, product and metabolite inhibition, and reversible phosphorylation (reviewed in refs. 13,17). Initial studies of the regulatory properties of plant mPDC have been conducted in vitro using preparations that had been purified 20-to 300-fold (18-21). Recently, we (2-4, 16) have developed a method for assaying mPDC activity in situ using purified intact pea seedling mitochondria. Using this method, several observations on the regulation by reversible phosphorylation have been clarified and extended (2-4). This report continues our investigation of the role of product inhibition in regulation of PDC activity within intact mitochondria.Much like mammalian and microbial PDCs, plant mPDCs

Expression of a yeast acetyl CoA hydrolase in the mitochondrion

“…Fernie, unpublished results). Moreover the well established regulatory loop by which low levels of mitochondrial acetyl CoA derepress the in vivo activity of the mitochondrial pyruvate dehydrogenase complex (Budde et al, 1991), are also in keeping with this hypothesis. The up-regulation of the plastidial acetyl CoA synthetase (and indeed the repression of photosynthesis) could also be mediated by an elevated acetate production in the transformants.…”

“…These findings are interesting on several levels. First, they are consistent with the presence of an acetyl CoA hydrolase activity within the mitochondria since lowering the mitochondrial acetyl coA would be expected to result in a higher activation state (Budde et al, 1991). When taken on its own this enhanced activation state could help to explain the increased cellular content of acetyl CoA since citrate synthase has a very low Km (acetylCoA) and would therefore compete effectively for the residual acetyl CoA in the mitochondria of the transformants.…”

“…Given that some of the above results were unexpected and that acetyl CoA has previously been demonstrated to be a potent regulator of pyruvate dehydrogenase (Tovar-Mendes et al, 1993;Budde et al, 1991;Rubin et al, 1978), we next determined the mitochondrial activity of pyruvate dehydrogenase and assayed its activation state. Since replicated mitochondrial preparations represent a significant amount of work we restricted this study to the wild type and two strongest acetyl CoA hydrolase expressors.…”

“…Bottom panel shows ribosomal RNA (rRNA). (Rubin et al, 1978;Budde et al, 1991;Tovar-Mendes et al, 2003). In these studies we uncovered a reduced activity of mitochondrial pyruvate dehydrogenase albeit present at a higher-activation state.…”

The final step of pantothenate biosynthesis in higher plants: cloning and characterization of pantothenate synthetase from Lotus japonicus and Oryza sativum (rice)

“…& ) is the common essential precursor to CoA and acyl carrier protein, which are cofactors required in many energy-yielding reactions and also by a large number of metabolic enzymes [1]. CoA is also involved in the regulation of key metabolic reactions such as those catalysed by pyruvate dehydrogenase [2]. Biosynthesis of pantothenate takes place in bacteria, fungi and plants only, while animals must obtain it from their diet [3].…”