Kinetic methods for the study of the enzyme systems of beta-oxidation.
Kinetic methods for studying the reactions of the "general" fatty acyl CoA dehydrogenase under three sets of substrate and enzyme concentration conditions have been developed. The reaction of butyryl-CoA and electron transfer flavoprotein (ETF) can be studied either under steady-state conditions with enzyme at catalytic concentration or under single-turnover conditions with enzyme in excess. Under the latter conditions, acyl-CoA dehydrogenase acts both as a catalyst and an ultimate electron-transfer acceptor. The reductive half-reaction of butyryl-CoA and enzyme can also be studied in a separate kinetic experiment. Comparison of the pH dependences of the rate constants and isotope effects of the steady-state reaction of butyryl-CoA and ETF with the same parameters for the reductive half-reaction is consistent with a mechanism involving transfer of electrons from butyryl-CoA to ETF within a ternary complex. An alternative mechanism in which the reductive half-reaction takes place prior to the binding and reaction of ETF seems unlikely because the pH 8.5 isotope effect on the reductive half-reaction is much larger than that on the complete reaction in spite of the fact that the rates of the reactions are comparable. The pH dependence of the Km for substrate and KI for inhibitor is consistent with a mechanism for transfer of electrons within the ternary complex which involves protonation of the C = O group of substrates. The protonation labilizes the C-2 proton and base catalysis of the removal of the C-2 proton results in the production of the active enzyme-substrate species, namely the C-2 anion of substrate.
D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases; D044926:Butyryl-CoA Dehydrogenase; D002384:Catalysis; D044927:Electron-Transferring Flavoproteins; D006863:Hydrogen-Ion Concentration; D007506:Iron-Sulfur Proteins; D007700:Kinetics; D008956:Models, Chemical; D009097:Multienzyme Complexes; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D000587:Oxidoreductases Acting on CH-NH Group Donors; D013379:Substrate Specificity
D044927:Electron-Transferring Flavoproteins; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; D010088:Oxidoreductases; D044944:Acyl-CoA Dehydrogenases; D044926:Butyryl-CoA Dehydrogenase; D042964:Acyl-CoA Dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors; C023120:electron-transferring-flavoprotein dehydrogenase