Department of Chemistry, Georgia State University, Atlanta, GA 30302-4098, USA.
Arch Biochem Biophys. 2010 Jul;499(1-2):1-5. doi: 10.1016/j.abb.2010.04.024. Epub 2010 May 4.
In the active site of choline oxidase, Glu312 participates in binding the trimethylammonium group of choline, thereby positioning the alcohol substrate properly for efficient hydride transfer to the enzyme-bound flavin. Previous studies have shown that substitution of Glu312 with aspartate results in a perturbed mechanism of hydride transfer, with a 260-fold decrease in the rate associated with the mutation. Here, the reaction of alcohol oxidation catalyzed by the Glu312Asp enzyme has been investigated with 3-hydroxypropyl-trimethylamine (3-HPTA), a choline analogue with an extra methylene, as substrate. The results of the kinetic investigation using steady state and rapid reaction approaches showed that the impaired ability of the Glu312Asp enzyme to catalyze a hydride transfer reaction can be effectively, but not completely, rescued in the presence of an extra methylene group on the substrate that compensates for the equivalent shortening of the side chain on residue 312. This observation is consistent with choline oxidase having evolved to optimally catalyze the oxidation of choline.
在胆碱氧化酶的活性部位，Glu312 参与结合胆碱的三甲铵基团，从而使醇底物正确定位，以有效地将氢化物转移到酶结合的黄素上。先前的研究表明，用天冬氨酸取代 Glu312 会导致氢化物转移机制受到干扰，突变相关的速率降低了 260 倍。在这里，使用 3-羟丙基三甲胺（3-HPTA）作为底物，研究了 Glu312Asp 酶催化的醇氧化反应。使用稳态和快速反应方法进行的动力学研究结果表明，Glu312Asp 酶催化氢化物转移反应的能力受损，可以在底物上额外增加一个亚甲基基团，有效地（但不是完全地）得到挽救，从而补偿残基 312 上侧链的等效缩短。这一观察结果与胆碱氧化酶的进化以最佳方式催化胆碱氧化是一致的。
