Husain M, Massey V
J Biol Chem. 1979 Jul 25;254(14):6657-66.
p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens is a NADPH-dependent, FAD-containing monooxygenase catalyzing the hydroxylation of p-hydroxybenzoate to form 3,4-dihydroxybenzoate in the presence of NADPH and molecular oxygen. The mechanism of this three-substrate reaction was investigated in detail at pH 6.6, 4 degrees C, by steady state kinetics, stopped flow spectrophotometry, and equilibrium binding experiments. The initial velocity patterns are consistent with a ping-pong type mechanism which involves two ternary complexes between the enzyme and substrates. The first ternary complex is formed by random addition of p-hydroxybenzoate and NADPH to the enzyme, followed by the release of the first product (NADP+). The reduced enzyme . p-hydroxybenzoate complex now reacts with oxygen, the third substrate, to form the second ternary complex. The enzyme-bound p-hydroxybenzoate then reacts with the activated oxygen to give 3,4-dihydroxybenzoate which is released regenerating the oxidized enzyme for the next cycle. The binding of p-hydroxybenzoate to the oxidized enzyme to form a 1:1 complex causes large, characteristic spectral perturbations and fluorescence quenching. The dissociation constant for the enzyme . substrate complex was obtained by titrations in which absorbance and/or fluorescence quenching was measured. The binding constants of NADPH to the enzyme with and without p-hydroxybenzoate were determined kinetically by measuring the rate of reduction of the enzyme at different concentrations of NADPH. The reduction of the enzyme proceeds extremely slowly in the absence of p-hydroxybenzoate. The presence of the substrate causes a dramatic stimulation (140,000-fold) in the rate of enzyme reduction. The anaerobic reduction of the enzyme by NADPH in the presence of p-hydroxybenzoate produces a transient charge-transfer intermediate. On the basis of the proposed mechanism, the dissociation constants for p-hydroxybenzoate and NADPH as well as the Michaelis constants for all the three substrates were calculated from the initial velocity data. The agreement obtained between various kinetic parameters from the initial rate measurements and those calculated from the individual rate constants determined in rapid reactions, strongly supports the proposed mechanism for the p-hydroxybenzoate hydroxylase reaction.
来自荧光假单胞菌的对羟基苯甲酸羟化酶（EC 1.14.13.2）是一种依赖NADPH、含FAD的单加氧酶，在NADPH和分子氧存在的情况下，催化对羟基苯甲酸的羟化反应生成3,4-二羟基苯甲酸。在pH 6.6、4℃条件下，通过稳态动力学、停流分光光度法和平衡结合实验，详细研究了这种三底物反应的机制。初始速度模式符合乒乓型机制，该机制涉及酶与底物之间的两个三元复合物。第一个三元复合物是由对羟基苯甲酸和NADPH随机添加到酶上形成的，随后释放出第一个产物（NADP⁺）。还原型酶·对羟基苯甲酸复合物现在与第三种底物氧气反应，形成第二个三元复合物。与酶结合的对羟基苯甲酸随后与活化的氧反应生成3,4-二羟基苯甲酸，该产物被释放出来，使氧化型酶再生以进行下一个循环。对羟基苯甲酸与氧化型酶结合形成1:1复合物会引起大的、特征性的光谱扰动和荧光猝灭。通过测量吸光度和/或荧光猝灭的滴定法获得酶·底物复合物的解离常数。通过测量不同NADPH浓度下酶的还原速率，动力学测定了NADPH在有和没有对羟基苯甲酸存在时与酶的结合常数。在没有对羟基苯甲酸的情况下，酶的还原极其缓慢。底物的存在会使酶还原速率显著提高（140,000倍）。在对羟基苯甲酸存在下，NADPH对酶的厌氧还原产生一个瞬态电荷转移中间体。根据提出的机制，从初始速度数据计算出对羟基苯甲酸和NADPH的解离常数以及所有三种底物的米氏常数。从初始速率测量得到的各种动力学参数与从快速反应中测定的各个速率常数计算得到的参数之间的一致性，有力地支持了所提出的对羟基苯甲酸羟化酶反应机制。
