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

Abstract

1. 1. Pig liver glycolic acid oxidase (glycolate: O 2 oxidoreductase, EC 1.1.3.1) can function with either 2,6-dichlorophenolindophenol (DCIP) (ν max = 1250 moles of glycolate per mole of flavin per min) or O 2 (ν 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 K i 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 α-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.