Effect of anions on the catalytic activity of pig liver glycolic acid oxidase

1. 1. Pig liver glycolic acid oxidase (glycolate: O2 oxidoreductase, EC 1.1.3.1) can function with either 2,6-dichlorophenolindophenol (DCIP) ([nu]max = 1250 moles of glycolate per mole of flavin per min) or O2 ([nu]max = 620 moles of glycolate per mole of flavin per min)9,10 as the electron acceptor.2. 2. Straight-chain monocarboxylic acids are non-competitive inhibitors of this enzyme with either glycolate or glyoxalate as the variable substrate and DCIP as the electron acceptor. Dicarboxylic acids are competitive inhibitors when glycolate is the variable substrate but they are non-competitive inhibitors when glyoxalate is the variable substrate. Phosphate and arsenate cause enzyme activation.3. 3. The binding affinity of the enzyme for monocarboxylic acids is proportional to the number of carbon atoms in the alkyl residue (n), and a straight line is obtained when log Ki is plotted versus n. These results suggest that electrostatic and hydrophobic forces, associated with a positive charge and a hydrophobic region at the active site, contribute to the binding affinity of monocarboxylic acids.4. 4. The binding affinity of the enzyme for oxalate is surprisingly high when compared with monocarboxylic acids. A decreased binding affinity is observed for other dicarboxylic acids as the number of carbon atoms increases. These results suggest that the complex of the enzyme with oxalate involves electrostatic interaction of both carboxylate groups of oxalate with two adjacent positively charged groups at the active site. A similar bidentate complex may explain the binding affinity of the enzyme for [alpha]-hydroxy acid substrates.5. 5. Conditions under which non-competitive inhibition can approximate competitive inhibition are discussed to account for differences in the inhibition patterns observed when glycolate and glyoxalate are used as the variable substrate.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33686/1/0000198.pd

Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS.

Ischaemia-reperfusion injury occurs when the blood supply to an organ is disrupted and then restored, and underlies many disorders, notably heart attack and stroke. While reperfusion of ischaemic tissue is essential for survival, it also initiates oxidative damage, cell death and aberrant immune responses through the generation of mitochondrial reactive oxygen species (ROS). Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion. Here we develop a comparative in vivo metabolomic analysis, and unexpectedly identify widely conserved metabolic pathways responsible for mitochondrial ROS production during ischaemia reperfusion. We show that selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia in a range of tissues and is responsible for mitochondrial ROS production during reperfusion. Ischaemic succinate accumulation arises from reversal of succinate dehydrogenase, which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. After reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo ischaemia-reperfusion injury in murine models of heart attack and stroke. Thus, we have identified a conserved metabolic response of tissues to ischaemia and reperfusion that unifies many hitherto unconnected aspects of ischaemia-reperfusion injury. Furthermore, these findings reveal a new pathway for metabolic control of ROS production in vivo, while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation after subsequent reperfusion is a potential therapeutic target to decrease ischaemia-reperfusion injury in a range of pathologies