On the catalytic role of the conserved active site residue His466 of choline oxidase
On the catalytic role of the conserved active site residue His466 of choline oxidase
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DOI：
10.1021/bi048056j
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发表时间：
2005-01-25
期刊：
影响因子：
2.9
通讯作者：
Gadda, G
中科院分区：
文献类型：
--
作者：
Ghanem, M;Gadda, G
醇氧化成醛是由许多黄素依赖性酶催化的，这些酶被归类为葡萄糖-甲醇-胆碱氧化还原酶超家族。这些酶在它们的底物结合域中表现出很少的序列相似性，但共享高度保守的催化位点，这表明它们的底物的氧化具有相似的活化机制。在这项研究中，完全保守的组氨酸残基在位置466的胆碱氧化酶被替换为丙氨酸残基的定点诱变和生化，光谱和机械特性的产生的CHO-H466 A突变酶的特点。CHO-H466 A显示以胆碱为底物的k（cat）和k（cat）/K-m值比野生型酶的值低60倍和1000倍，而氧的k（cat）/k（m）值不受影响，表明His（466）参与醇底物的氧化，但不参与氧的还原。用丙氨酸取代His 466显著影响黄素的微环境，如CHO-H466 A与亚硫酸盐和连二亚硫酸盐的改变的行为所示。与这一结论一致，在pH 7下测定CHO-H466 A的催化活性酶-产物复合物中双电子转移的中点还原电位为+106 mV，比野生型酶的中点还原电位负25 mV。CHO-H466 A中的酶活性可以用外源性咪唑鎓而不是咪唑部分地拯救，这与组氨酸的质子化形式发挥催化作用一致。甘氨酸甜菜碱抑制的pH曲线、N（3）-黄素位点的去质子化和胆碱的kcat/Km值都显示出它们的pK（a）值显著上移，与活性位点极性的变化一致。最后，同位素标记的底物和溶剂的动力学同位素效应表明，组氨酸丙氨酸取代影响底物OH和CH键裂解的时间，与去除羟基质子一致，与突变酶中的氢化物转移一致。总之，本研究中呈现的结果表明，在胆碱氧化酶中，His（466）调节酶结合黄素的亲电性和活性位点的极性，并有助于稳定胆碱氧化为甜菜碱醛的过渡态。
The oxidation of alcohols to aldehydes is catalyzed by a number of flavin-dependent enzymes, which have been grouped in the glucose-methanol-choline oxidoreductase enzyme superfamily. These enzymes exhibit little sequence similarity in their substrates binding domains, but share a highly conserved catalytic site, suggesting a similar activation mechanism for the oxidation of their substrates. In this study, the fully conserved histidine residue at position 466 of choline oxidase was replaced with an alanine residue by site-directed mutagenesis and the biochemical, spectroscopic, and mechanistic properties of the resulting CHO-H466A mutant enzyme were characterized. CHO-H466A showed k(cat), and k(cat)/K-m values with,choline as substrate that were 60- and 1000-fold lower than the values for the wild-type enzyme, while the k(cat)/k(m) value for oxygen was unaffected, suggesting the involvement of His(466) in the oxidation of the alcohol substrate but not in the reduction of oxygen. Replacement of His466 with alanine significantly affected the microenvironment of the flavin, as indicated by the altered behavior of CHO-H466A with sulfite and dithionite. In agreement with this conclusion, a midpoint reduction potential of +106 mV for the two-electron transfer in the catalytically competent enzyme-product complex was determined at pH 7 for CHO-H466A, which was similar to25 mV more negative than that of the wild-type enzyme. Enzymatic activity in CHO-H466A could be partially rescued with exogenous imidazolium, but not imidazole, consistent with the protonated form of histidine exerting a catalytic role. pH profiles for glycine betaine inhibition, the deprotonation of the N(3)-flavin locus, and the kcat/Km value for choline all showed a significant shift upward in their pK(a) values, consistent with a change in the polarity of the active site. Finally, kinetic isotope effects with isotopically labeled substrate and solvent indicated that the histidine to alanine substitution affected the timing of substrate OH and CH bond cleavages, consistent with removal of the hydroxyl proton being concerted with hydride transfer in the mutant enzyme. All taken together, the results presented in this study suggest that in choline oxidase, His(466) modulates the electrophilicity of the enzyme-bound flavin and the polarity of the active site, and contributes to the stabilization of the transition state for the oxidation of choline to betaine aldehyde.