Hayashi H, Mizuguchi H, Kagamiyama H
Department of Biochemistry, Osaka Medical College, Takatsuki, Japan.
Biochemistry. 1998 Oct 27;37(43):15076-85. doi: 10.1021/bi981517e.
In aspartate aminotransferase, pyridoxal 5'-phosphate (PLP) forms a Schiff base with the epsilon-amino group of Lys258 (internal aldimine). The internal aldimine has a pKa value of 6.8. Binding of a substrate amino acid to the enzyme yields the Michaelis complex, in which PLP still forms the internal aldimine with Lys258. This is followed by a transaldimination process to form a Schiff base of PLP with the alpha-amino group of substrates (external aldimine). Kinetic analysis of the spectral changes during the reaction of the enzyme with a substrate analogue 2-methylaspartate showed that the aldimine is 6.4-8.6% protonated in the Michaelis complex and 32-43% in the external aldimine. The bases that accept protons from the aldimines are considered to be the substrate alpha-amino group in the Michaelis complex and the epsilon-amino group of Lys258 in the external aldimine. Therefore, the intrinsic pKa value of the aldimine is expected to increase over a range of 3 during transformation from the unliganded enzyme (pKa = 6.8) to the Michaelis complex (pKa = 8.8) and the external aldimine (pKa > 10). When the Lys258 side chain of the internal aldimine was "cleaved" by the construction of an enzyme in which Lys258 was replaced by Ala and the aldimine was reconstituted with methylamine, the pKa of the internal aldimine was increased to 9.6. This indicates that the low pKa value of the internal aldimine of the unliganded enzyme is provided by the side chain of Lys258 which destabilizes the planar conformation of the aldimine suitable for protonation. This strained conformation is partially relaxed in the Michaelis complex, and the pKa is moderately increased. On formation of the external aldimine, Lys258 is released and the aldimine is fixed to a near planar conformation and has a high pKa value. Thus, the aldimine pKa is modulated by a mechanism that exploits the conformational differences between the intermediate structures. The strain of the protonated internal aldimine is interpreted to enhance the catalytic ability of the enzyme by increasing the energy level of the free enzyme plus substrate at neutral pH relative to the transition state.
在天冬氨酸转氨酶中，磷酸吡哆醛（PLP）与赖氨酸258的ε-氨基形成席夫碱（内部醛亚胺）。内部醛亚胺的pKa值为6.8。底物氨基酸与酶结合产生米氏复合物，其中PLP仍与赖氨酸258形成内部醛亚胺。随后发生转醛亚胺化过程，形成PLP与底物α-氨基的席夫碱（外部醛亚胺）。对酶与底物类似物2-甲基天冬氨酸反应过程中光谱变化的动力学分析表明，醛亚胺在米氏复合物中的质子化程度为6.4 - 8.6%，在外部醛亚胺中的质子化程度为32 - 43%。被认为从醛亚胺接受质子的碱在米氏复合物中是底物α-氨基，在外部醛亚胺中是赖氨酸258的ε-氨基。因此，预计醛亚胺的固有pKa值在从未结合配体的酶（pKa = 6.8）转变为米氏复合物（pKa = 8.8）和外部醛亚胺（pKa > 10）的过程中会在3的范围内增加。当通过构建一种将赖氨酸258替换为丙氨酸且用甲胺重建醛亚胺的酶来“切断”内部醛亚胺的赖氨酸258侧链时，内部醛亚胺的pKa增加到9.6。这表明未结合配体的酶的内部醛亚胺的低pKa值是由赖氨酸258的侧链提供的，该侧链使适合质子化的醛亚胺平面构象不稳定。这种紧张构象在米氏复合物中部分松弛，pKa适度增加。在形成外部醛亚胺时，赖氨酸258被释放，醛亚胺固定为接近平面的构象并具有高pKa值。因此，醛亚胺pKa是通过一种利用中间结构之间构象差异的机制来调节的。质子化内部醛亚胺的张力被解释为通过在中性pH下相对于过渡态提高游离酶加底物的能量水平来增强酶的催化能力。
