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 re ducing equivalents to electron-transferring flavoprotein. The binding of normal substrates profoundly suppresses the reactivity of the reduc ed enzyme toward molecular oxygen, whereas the oxidase reaction become s significant using thioesters such as indolepropionyl-CoA (IP-CoA) an d 4-(dimethylamino)-3-phenylpropionyl-CoA (DP-CoA). Steady-state and s topped-flow studies with IP-CoA led to a kinetic model of the oxidase reaction in which only the free reduced enzyme reacts with oxygen (Joh nson, J. K., Kumar, N. R., and Srivastava, D. K. (1994) Biochemistry 3 3, 4738-4744). We have tested their proposal with IP-CoA and DP-CoA. T he dependence of the oxidase reaction on oxygen concentration is bipha sic with a major low affinity phase incompatible with a model predicti ng a simple K-m for oxygen of 3 mu M. If only free reduced enzyme reac ts with oxygen, increasing IP-CoA would show strong substrate inhibiti on because it binds tightly to the reduced enzyme. Experimentally, IP- CoA shows simple saturation kinetics. The Glu376-Gln mutant of the med ium chain dehydrogenase allows the oxygen reactivity of complexes of t he reduced enzyme with IP-CoA and the corresponding product indoleacry loyl-CoA (IA-CoA) to be characterized without the subsequent redox equ ilibration that complicates analysis of the oxidase kinetics of the na tive enzyme. in sum, these data suggest that when bulky, nonphysiologi cal substrates are employed, multiple reduced enzyme species react wit h molecular oxygen. The relatively high oxidase activity of the short chain acyl-CoA dehydrogenase from the obligate anaerobe Megasphaera el sdenii was studied by rapid reaction kinetics of wild-type and the Glu 367-Gln mutant using butyryl-, crotonyl-, and 2-aza-butyryl-CoA thioes ters. In marked contrast to those of the mammalian dehydrogenase, comp lexes of the reduced bacterial enzyme with these ligands react with mo lecular oxygen at rates similar to those of the free protein. Evolutio nary and mechanistic aspects of the suppression of oxygen reactivity i n the acyl-CoA dehydrogenases are discussed.