Oxidase activity of the acyl-CoA dehydrogenases.
The medium chain acyl-CoA dehydrogenase catalyzes the flavin-dependent oxidation of a variety of acyl-CoA thioesters with the transfer of reducing equivalents to electron-transferring flavoprotein. The binding of normal substrates profoundly suppresses the reactivity of the reduced enzyme toward molecular oxygen, whereas the oxidase reaction becomes significant using thioesters such as indolepropionyl-CoA (IP-CoA) and 4-(dimethylamino)-3-phenylpropionyl-CoA (DP-CoA). Steady-state and stopped-flow studies with IP-CoA led to a kinetic model of the oxidase reaction in which only the free reduced enzyme reacts with oxygen (Johnson, J. K., Kumar, N. R., and Srivastava, D. K. (1994) Biochemistry 33, 4738-4744). We have tested their proposal with IP-CoA and DP-CoA. The dependence of the oxidase reaction on oxygen concentration is biphasic with a major low affinity phase incompatible with a model predicting a simple Km for oxygen of 3 microM. If only free reduced enzyme reacts with oxygen, increasing IP-CoA would show strong substrate inhibition because it binds tightly to the reduced enzyme. Experimentally, IP-CoA shows simple saturation kinetics. The Glu376-Gln mutant of the medium chain dehydrogenase allows the oxygen reactivity of complexes of the reduced enzyme with IP-CoA and the corresponding product indoleacryloyl-CoA (IA-CoA) to be characterized without the subsequent redox equilibration that complicates analysis of the oxidase kinetics of the native enzyme. In sum, these data suggest that when bulky, nonphysiological substrates are employed, multiple reduced enzyme species react with molecular oxygen. The relatively high oxidase activity of the short chain acyl-CoA dehydrogenase from the obligate anaerobe Megasphaera elsdenii was studied by rapid reaction kinetics of wild-type and the Glu367-Gln mutant using butyryl-, crotonyl-, and 2-aza-butyryl-CoA thioesters. In marked contrast to those of the mammalian dehydrogenase, complexes of the reduced bacterial enzyme with these ligands react with molecular oxygen at rates similar to those of the free protein. Evolutionary and mechanistic aspects of the suppression of oxygen reactivity in the acyl-CoA dehydrogenases are discussed.
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.
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DuPlessis
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Pellett
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Stankovich
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Thorpe
D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D003198:Computer Simulation; D006801:Humans; D007668:Kidney / Q000201:enzymology; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine; D045854:Veillonellaceae / Q000201:enzymology / Q000235:genetics
D011994:Recombinant Proteins; D010088:Oxidoreductases; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase; D010100:Oxygen