Bohne C, MacDonald I D, Dunford H B
J Biol Chem. 1987 Mar 15;262(8):3572-8.
Elementary reactions have been studied quantitatively in the complex overall process catalyzed by horseradish peroxidase whereby isobutyraldehyde and molecular oxygen react to form triplet state acetone and formic acid. The rate constant for the reaction of the enol form of isobutyraldehyde with compound I of peroxidase is (8 +/- 1) X 10(6) M-1 s-1 and with compound II (1.3 +/- 0.3) X 10(6) M-1 s-1. Neither the enolate anion nor the keto form is reactive. The reactivity of enols with peroxidase parallels that of unionized phenols and a common mechanism is proposed. The overall catalyzed reaction of isobutyraldehyde and oxygen consists of an initial burst followed by a steady state phase. The burst is caused by the following sequence: 1) an initial high yield of compound I is formed from reaction of native enzyme with the autoxidation product of isobutyraldehyde, a peracid and 2) compound I rapidly depletes the equilibrium pool of enol which is present. After this burst a steady state phase is observed in which the rate-limiting step is the conversion of the keto to the enol form of the aldehyde catalyzed by phosphate buffer. The rate constant for the keto form reacting with phosphate is (8.7 +/- 0.6) X 10(-5) M-1 s-1. All constants were measured in dilute aqueous ethanol at 35 degrees C, pH 7.4, and ionic strength 0.67 M. Both the initial burst of light and the steady state emission from triplet acetone can be observed with the naked eye. Since the magnitude of the burst is a measure of the equilibrium amount of enol, the keto-enol equilibrium constant is readily calculated and hence also the rate constant for conversion of enol to keto. The keto-enol equilibrium constant is unaffected by phosphate which therefore acts as a true catalyst.
在辣根过氧化物酶催化的复杂整体过程中，对基本反应进行了定量研究，在此过程中异丁醛与分子氧反应生成三重态丙酮和甲酸。异丁醛烯醇形式与过氧化物酶化合物I反应的速率常数为(8±1)×10⁶ M⁻¹ s⁻¹，与化合物II反应的速率常数为(1.3±0.3)×10⁶ M⁻¹ s⁻¹。烯醇负离子和酮形式均无反应活性。烯醇与过氧化物酶的反应活性与未电离酚类的反应活性相似，并提出了一种共同的机制。异丁醛与氧气的整体催化反应包括一个初始爆发阶段，随后是一个稳态阶段。爆发是由以下序列引起的：1) 天然酶与异丁醛的自氧化产物（一种过酸）反应，最初高产率地形成化合物I；2) 化合物I迅速耗尽存在的烯醇平衡池。在这个爆发阶段之后，观察到一个稳态阶段，其中限速步骤是由磷酸盐缓冲液催化的醛从酮形式向烯醇形式的转化。酮形式与磷酸盐反应的速率常数为(8.7±0.6)×10⁻⁵ M⁻¹ s⁻¹。所有常数均在35℃、pH 7.4和离子强度0.67 M的稀乙醇水溶液中测量。三重态丙酮的初始光爆发和稳态发射都可以用肉眼观察到。由于爆发的大小是烯醇平衡量的一种度量，因此很容易计算酮-烯醇平衡常数，从而也能计算出烯醇向酮转化的速率常数。酮-烯醇平衡常数不受磷酸盐的影响，因此磷酸盐起到了真正催化剂的作用。
