Beaty N B, Ballou D P
J Biol Chem. 1981 May 10;256(9):4619-25.
The reactions of reduced forms of liver microsomal FAD-containing monooxygenase both with oxygen and with several oxygenatable substrates have been investigated using rapid reaction techniques. It was first shown that the pH optimum was at about 8.8 and that at pH values below 8 the stoichiometry was very nearly 1:1:1 for NADPH/(cysteamine or methimazole)/oxygen consumption, with only traces of H2O2 forming. Low temperatures (approximately 4 degrees C) were employed to better resolve kinetic intermediates occurring in the reaction. It was shown that in the absence of NADP+, substrate has no effect on the rate of reoxidation of reduced enzyme by O2. However, in the presence of NADP+ and the absence of substrate, the reduced enzyme reacts with O2 to form a remarkably stable C(4a)-hydroperoxyflavin species. This occurs in a reaction that saturates with respect to O2, implying the formation of a reversible complex with oxygen prior to the formation of the hydroperoxyflavin. This has been predicted by the steady state kinetics (Poulsen, L. L., and Ziegler, D. M. (1979) J. Biol. Chem. 254, 6449-6455). In the presence of NADP+, substrate increases the rate of reoxidation (by destabilizing the hydroperoxyflavin), but the rate is much slower than the reaction of reduced enzyme with O2 in the absence of NADP+. The stability of the hydroperoxyflavin permitted its preparation on the stopped flow apparatus for subsequent reaction with various substrates. This reaction involved the rapid formation of a species with a new spectrum which was indicative of a C(4a)-substituted flavin. Since this new species converts to oxidized flavin at about 2 min-1, which is equivalent to Vmax, it must be the rate-limiting step in the catalytic reaction. This new spectrum is likely due either to a substrate-hydroperoxyflavin species or to a product-hydroxyflavin species.
利用快速反应技术研究了肝脏微粒体含黄素腺嘌呤二核苷酸单加氧酶还原形式与氧气以及几种可氧化底物的反应。首先发现最适pH约为8.8，且在pH值低于8时，NADPH/（半胱胺或甲巯咪唑）/氧气消耗的化学计量比非常接近1:1:1，仅形成微量的过氧化氢。采用低温（约4℃）以更好地解析反应中出现的动力学中间体。结果表明，在没有NADP⁺的情况下，底物对还原酶被O₂再氧化的速率没有影响。然而，在存在NADP⁺且没有底物的情况下，还原酶与O₂反应形成一种非常稳定的C(4a)-氢过氧黄素物种。这一反应在O₂饱和时发生，意味着在氢过氧黄素形成之前与氧气形成了可逆复合物。这已由稳态动力学预测（Poulsen, L. L., and Ziegler, D. M. (1979) J. Biol. Chem. 254, 6449 - 6455）。在存在NADP⁺的情况下，底物会增加再氧化速率（通过使氢过氧黄素不稳定），但该速率比在没有NADP⁺时还原酶与O₂的反应慢得多。氢过氧黄素的稳定性使其能够在停流装置上制备，以便随后与各种底物反应。该反应涉及快速形成一种具有新光谱的物种，这表明是一种C(4a)-取代黄素。由于这种新物种以约2 min⁻¹的速率转化为氧化黄素，这相当于Vmax，所以它一定是催化反应中的限速步骤。这种新光谱可能是由于底物 - 氢过氧黄素物种或产物 - 羟基黄素物种所致。
