Gadda Giovanni
Department of Chemistry, Georgia State University, P.O. Box 4098, Atlanta, GA 30302-4098, USA.
Biochim Biophys Acta. 2003 Mar 21;1646(1-2):112-8. doi: 10.1016/s1570-9639(03)00003-7.
Choline oxidase catalyzes the four-electron oxidation of choline to glycine-betaine, with betaine-aldehyde as intermediate and molecular oxygen as primary electron acceptor. The enzyme is capable of accepting betaine-aldehyde as a substrate, allowing the investigation of the reaction mechanism for both the conversion of choline to the aldehyde intermediate and of betaine-aldehyde to glycine-betaine. The steady state kinetic mechanism has been determined at pH 7 with choline and betaine-aldehyde as substrate to be sequential, consistent with oxygen reacting with the reduced enzyme before release of betaine-aldehyde or glycine-betaine, respectively. A K(m) value < or =20 microM has been estimated for betaine-aldehyde based on the kinetic pattern with a y-intercept seen in a plot of 1/rate versus 1/[oxygen]. The kinetic data suggest that betaine-aldehyde predominantly remains bound at the active site during turnover of the enzyme with choline. In agreement with such a conclusion, less than 10% betaine-aldehyde has been found in the reaction mixture under enzymatic turnover with saturating concentrations of choline. The k(cat) values were 6.4+/-0.3 and 15.3+/-2.5 s(-1) for choline and betaine-aldehyde, respectively, suggesting that a kinetic step in the oxidation of choline to the aldehyde intermediate must be partially rate-limiting for catalysis. Cleavage of the CH bond of choline as being partially rate-limiting for catalysis is discussed.
胆碱氧化酶催化胆碱进行四电子氧化反应生成甘氨酸甜菜碱，甜菜碱醛为中间产物，分子氧作为主要电子受体。该酶能够接受甜菜碱醛作为底物，从而可以研究胆碱转化为醛中间体以及甜菜碱醛转化为甘氨酸甜菜碱的反应机制。在pH 7条件下，以胆碱和甜菜碱醛为底物测定了稳态动力学机制，结果表明该反应是顺序反应，这与氧气分别在甜菜碱醛或甘氨酸甜菜碱释放之前与还原态酶反应一致。根据1/速率对1/[氧气]作图时的y轴截距所呈现的动力学模式，估计甜菜碱醛的K(m)值≤20 μM。动力学数据表明，在酶与胆碱周转过程中，甜菜碱醛主要保持结合在活性位点上。与此结论一致的是，在饱和浓度胆碱的酶促周转反应混合物中，发现甜菜碱醛含量不到10%。胆碱和甜菜碱醛的k(cat)值分别为6.4±0.3和15.3±2.5 s(-1)，这表明胆碱氧化为醛中间体的动力学步骤必定是催化反应的部分限速步骤。文中讨论了胆碱中C-H键的断裂作为催化反应部分限速步骤的情况。
