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

详细研究了芳香L氨基酸脱羧酶、天冬氨酸转氨酶和芳香族氨基酸转氨酶三种依赖于5‘-磷酸吡哆醛的酶的质子转移过程。具有去质子化a-氨基的底物氨基酸优先与AADC结合，但带有质子化a-氨基的底物也可以结合较小的程度。在米氏络合物中，底物α-胺基团上的质子由于与质子化PLP席夫碱的静电排斥而解离。在AAT和ART中，发现*Re是质子化PLP席夫碱中的构象应变，它在催化过程中相继释放。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，静电相互作用在调节活性中心催化基团的pKa方面的作用都小于Schiff碱菌株。总而言之，在AADC中，底物和酶之间的菌株被认为增加了k_&lt；cat&gt；值，而在AAT和ART中，酶蛋白固有的菌株以包括过渡态在内的中间状态释放，增加了k_&lt；cat&gt；/K_m值。这种机制在包括“质子数”轴作为坐标之一的三维能量分布中被最清楚地理解。这些轮廓清楚地解释了质子转移的驱动力，表明pK_a的概念在催化能量学中具有次要意义。

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.