DuPlessis E R, Pellett J, Stankovich M T, Thorpe C
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.
Biochemistry. 1998 Jul 21;37(29):10469-77. doi: 10.1021/bi980767s.
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.
中链酰基辅酶A脱氢酶催化多种酰基辅酶A硫酯的黄素依赖性氧化反应，将还原当量转移至电子传递黄素蛋白。正常底物的结合会显著抑制还原型酶与分子氧的反应活性，而使用吲哚丙酰辅酶A（IP-CoA）和4-（二甲基氨基）-3-苯基丙酰辅酶A（DP-CoA）等硫酯时，氧化酶反应则变得显著。对IP-CoA进行的稳态和停流研究得出了氧化酶反应的动力学模型，其中只有游离的还原型酶与氧反应（约翰逊，J.K.，库马尔，N.R.，和斯里瓦斯塔瓦，D.K.（1994年）《生物化学》33卷，4738 - 4744页）。我们用IP-CoA和DP-CoA对他们的提议进行了验证。氧化酶反应对氧浓度的依赖性呈双相，主要的低亲和力相与预测氧的简单米氏常数为3微摩尔的模型不相符。如果只有游离的还原型酶与氧反应，增加IP-CoA会表现出强烈的底物抑制作用，因为它与还原型酶紧密结合。实验表明IP-CoA呈现简单的饱和动力学。中链脱氢酶的Glu376-Gln突变体能够对还原型酶与IP-CoA及相应产物吲哚丙烯酰辅酶A（IA-CoA）形成的复合物的氧反应活性进行表征，而不会出现使天然酶氧化酶动力学分析复杂化的后续氧化还原平衡。总之，这些数据表明，当使用体积较大的非生理性底物时，多种还原型酶会与分子氧反应。通过对野生型和Glu367-Gln突变体使用丁酰、巴豆酰和2-氮杂丁酰辅酶A硫酯进行快速反应动力学研究，对专性厌氧菌埃氏巨球形菌的短链酰基辅酶A脱氢酶相对较高的氧化酶活性进行了研究。与哺乳动物脱氢酶形成显著对比的是，还原型细菌酶与这些配体形成的复合物与分子氧反应的速率与游离蛋白相似。文中还讨论了酰基辅酶A脱氢酶中氧反应活性抑制的进化和机制方面的问题。
