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The proton-transfer mechanism of the catalytic reactions of pyridoxal enzymes

吡哆醛酶催化反应的质子转移机制

基本信息

批准号：
11680641
负责人：
HAYASHI Hideyuki
金额：
$ 2.37万
依托单位：
Osaka Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

The proton-transfer processes have been studied in detail for three kinds of pyridoxal 5'-phosphate (PLP)-dependent enzymes, aromatic L-amine acid decarboxylase (AADC), aspartate aminotransferase (AAT), and aromatic amino acid aminotransferase (ART). The substrate amino acid with deprotonated a-amine group is preferentially bound to AADC, but the substrate with protonated a-amino group can also be bound to a lesser extent. In the Michaelis complex, the proton on the substrate a-amine group is dissociated because of the electrostatic repulsion with the protonated PLP Schiff base of AADC.In AAT and ART, it was found that *re is a conformational strain in the protonated PLP Schiff base, which is released successively during the course of catalysis. This mechanism is important for regulating the proton-transfer events in the catalytic process. In both AAT and ART electrostatic interactions play minor roles than the Schiff base strain in regulating the pK_a of the active site catalytic group. Altogether, in AADC, the strain between the substrate and the enzyme is considered to increase the k_<cat> value, whereas in AAT and ART, the strain inherent to the enzyme protein but is released in the intermediate including the transition state, increases the k_<cat>/K_m value. This mechanism is most clearly understood in the 3-dimensional energy profiles, which include the "proton number" axis as one of the coordinates. These profiles clearly explain the driving force of proton transfer, showing that the notion of pK_a has a secondary significance in the energetics of catalysis.

本文详细研究了5 ′-磷酸吡哆醛（PLP）依赖的芳香族L-氨基酸脱羧酶（AADC）、天冬氨酸氨基转移酶（AAT）和芳香族氨基酸氨基转移酶（ART）的质子转移过程。具有去质子化的α-氨基的底物氨基酸优先与AADC结合，但具有质子化的α-氨基的底物也可以在较小程度上结合。在米氏络合物中，底物α-氨基上的质子由于与AADC的质子化PLP Schiff碱的静电排斥而解离，在AAT和ART中，发现 *re是质子化PLP Schiff碱中的构象应变，在催化过程中连续释放。这一机制对于调节催化过程中的质子转移事件具有重要意义。在AAT和ART中，静电相互作用对活性中心催化基团pK_a的调节作用比Schiff碱小。总之，在AADC中，底物和酶之间的应变被认为增加了k_<cat>值，而在AAT和ART中，酶蛋白固有的应变但在包括过渡态的中间体中释放，增加了k_<cat>/K_m值。这一机制在三维能量分布图中得到了最清楚的理解，其中包括作为坐标之一的“质子数”轴。这些曲线清楚地解释了质子转移的驱动力，表明pK_a的概念在催化的能量学中具有次要意义。