Purification and characterization of pumpkin long-chain acyl-CoA oxidase

Pumpkin (Cucurbita sp.) long-chain acyl-CoA oxidase (ACOX) (EC 1.3.3.6) was purified to homogeneity by hydrophobic interaction, hydroxyapatite, affini ty, and anion exchange chromatographies. The purified isoenzyme is a dimeri c protein, consisting of two apparently identical 72-kDa subunits. The prot ein is exclusively localized in glyoxysomes. The enzyme catalyzes selective ly the oxidation of CoA esters of fatty acids with 12-18 C atoms and exhibi ts highest activity with C-14 fatty acids, but no activity with isobutyryl- CoA and isovaleryl-CoA (branched chain) or glutaryl-CoA (dicarboxylic). The enzyme is strongly inhibited by high concentrations of palmitoyl-CoA and w eakly inhibited by high concentration of myristoyl-CoA. It is also inhibite d by Triton X-100 at concentrations above 0.018% (w/v) the critical micella r concentration. The consequences of the substrate inhibition for the evalu ation of long-chain ACOX activity in plant tissues are discussed.