Yano T, Hinoue Y, Chen V J, Metzler D E, Miyahara I, Hirotsu K, Kagamiyama H
Department of Medical Chemistry, Osaka Medical College, Japan.
J Mol Biol. 1993 Dec 20;234(4):1218-29. doi: 10.1006/jmbi.1993.1672.
Asp222 of aspartate aminotransferase is an active-site residue which interacts with the pyridine nitrogen of the coenzyme, pyridoxal 5'-phosphate (PLP). The roles of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase have previously been explored by site-directed mutagenesis. These studies confirmed that a negatively charged residue at position 222 is essential for catalysis, but the reason for this remained speculative. In the present studies, the roles of Asp222 were clarified experimentally by analyzing the mutant D222A enzyme (Asp222 replaced by Ala) reconstituted with the coenzyme analog N(1)-methylated PLP (N-MePLP). Spectroscopic and kinetic analyses showed that Asp222 stabilizes the protonated N(1) of PLP, raising the pKa value of N(1) by more than five units, in the active site of AspAT. The positive charge at N(1) accelerates abstraction of the alpha-proton from the amino acid substrate, stabilizing the transition state by 1.4 to 4.5 kcal.mol-1 in the reaction with aspartate. X-ray crystallographic (2.0 A resolution) and CD spectroscopic studies suggest that the coenzyme analog is not held in a proper orientation within the active site of D222A (N-MePLP). This may account for the finding that the catalytic activity was recovered only partially by the reconstitution of D222A with N-MePLP. These results fully support the following postulated role of Asp222: the negative charge of Asp222 stabilizes the positive charge at N(1) of PLP and thereby enhances the function of PLP as an electron sink.
天冬氨酸转氨酶的Asp222是一个活性位点残基，它与辅酶磷酸吡哆醛（PLP）的吡啶氮相互作用。此前已通过定点诱变研究了Asp222在大肠杆菌天冬氨酸转氨酶催化机制中的作用。这些研究证实，222位带负电荷的残基对催化至关重要，但其原因仍属推测。在本研究中，通过分析用辅酶类似物N(1)-甲基化PLP（N-MePLP）重构的突变体D222A酶（Asp222被Ala取代），实验阐明了Asp222的作用。光谱和动力学分析表明，Asp222稳定了PLP的质子化N(1)，使AspAT活性位点中N(1)的pKa值提高了五个以上单位。N(1)处的正电荷加速了从氨基酸底物中提取α-质子的过程，在与天冬氨酸的反应中使过渡态稳定了1.4至4.5 kcal.mol-1。X射线晶体学（分辨率为2.0 Å）和圆二色光谱研究表明，辅酶类似物在D222A（N-MePLP）的活性位点内未处于正确的方向。这可能解释了用N-MePLP重构D222A仅部分恢复催化活性这一发现。这些结果充分支持了Asp222的以下假定作用：Asp222的负电荷稳定了PLP的N(1)处的正电荷，从而增强了PLP作为电子受体的功能。
