Reductive and oxidative half-reactions of morphinone reductase from Pseudomonas putida M10:: A kinetic and thermodynamic analysis
Reductive and oxidative half-reactions of morphinone reductase from Pseudomonas putida M10:: A kinetic and thermodynamic analysis
复制标题
DOI：
10.1021/bi980345i
复制
发表时间：
1998-05-19
期刊：
影响因子：
2.9
通讯作者：
Scrutton, NS
中科院分区：
文献类型：
--
作者：
Craig, DH;Moody, PCE;Scrutton, NS
采用多波长和单波长停流光谱法研究了吗啡酮还原酶（MR）与生理还原剂NADH和氧化底物可待因酮的反应。酶与NADH的还原分两个动力学可分解的步骤进行。在第一步中，被氧化的酶与NADH形成电荷转移中间体。该电荷转移配合物的特点是在长波长（540 ~ 650 nm）处吸光度增加，其形成速率依赖于底物浓度，在pH 7.0和5℃下由4.8 x 10(5) M-1 s（-1）的二阶速率常数控制。第二步，酶结合黄素被还原为二氢黄素形式。黄素的还原速率（在pH 7.0和5℃时为23.4 s(-1)）与底物浓度无关，并且在462 nm处观察到吸光度的单相下降。氧化半反应分三个动力学可分辨的步骤进行。第一个是由于还原酶-可待因酮电荷转移复合物的形成，并在长波长（约650 nm）下观察到。电荷转移络合物的形成速率依赖于可待酮浓度，在pH 7.0和5℃时由11.5 x 10(3) M-1 s（-1）的二阶速率常数控制。第二步代表黄素再氧化，在462 nm（吸收增加）和650 nm（吸收减少）处观察到，并以与可待酮浓度无关的速率（约45 s(-1)）进行。第三步观察到在462 nm处吸光度进一步小幅增加，并且以约2.5 s（-1）的速率进行。这一步很可能代表氢可酮从氧化酶中释放出来。通过分析还原半反应的温度依赖性，可以计算出电荷转移形成、电荷转移衰变（产生游离酶和底物）和电子向酶结合的FMN转移的熵和焓贡献，并构建了MR催化反应的部分能量谱。MR的反应方案和氧化还原性质与之前描述的密切相关的黄蛋白进行了比较。老黄酶。虽然确定了两种酶的共同特征，但在动力学和氧化还原特性方面存在显着差异。
The reaction of morphinone reductase (MR) with the physiological reductant NADH and the oxidizing substrate codeinone has been studied by multiple and single wavelength stopped-flow spectroscopy. Reduction of the enzyme with NADH proceeds in two kinetically resolvable steps. In the first step, the oxidized enzyme forms a charge-transfer intermediate with NADH. The charge-transfer complex is characterized by an increase in absorbance at long wavelength (540 to 650 nm), and its rate of formation is dependent on substrate concentration and is controlled by a second-order rate constant of 4.8 x 10(5) M-1 s(-1) at pH 7.0 and 5 degrees C. In the second step, the enzyme-bound flavin is reduced to the dihydroflavin form. The rate of flavin reduction (23.4 s(-1) at pH 7.0 and 5 degrees C) is independent of substrate concentration and is observed as a monophasic decrease in absorbance at 462 nm. The oxidative half-reaction proceeds in three kinetically resolvable steps. The first is due to the formation of a reduced enzyme-codeinone charge-transfer complex and is observed at long wavelength (about 650 nm). The rate of charge-transfer complex formation is dependent on codeinone concentration and is controlled by a second-order rate constant of 11.5 x 10(3) M-1 s(-1) at pH 7.0 and 5 degrees C. The second step represents flavin reoxidation and is observed at 462 (absorption increase) and 650 nm (absorption decrease) and progresses with a rate (about 45 s(-1)) which is independent of codeinone concentration. The third step is observed as a further small increase in absorbance at 462 nm and proceeds with a rate of about 2.5 s(-1). This step most likely represents hydrocodone release from the oxidized enzyme. Analysis of the temperature dependence of the reductive half-reaction has enabled calculation of the entropic and enthalpic contributions for charge-transfer formation, charge-transfer decay (yielding free enzyme and substrate), and electron transfer to the enzyme-bound FMN, and the construction of a partial energy profile for the reaction catalyzed by MR. The reaction scheme and redox properties of MR are compared with those described previously for the closely related flavoprotein, old yellow enzyme. Although common features are identified, there are notable differences in the kinetic and redox properties of the two enzymes.