<br>磷酸吡哆醛依赖性天冬氨酸氨基转移酶的光增强催化,Journal of the American Chemical Society

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磷酸吡哆醛依赖性天冬氨酸氨基转移酶的光增强催化
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2010-12-01 , DOI: 10.1021/ja107054x
Melissa P Hill ₁ , Elizabeth C Carroll , Mai C Vang , Trevor A Addington , Michael D Toney , Delmar S Larsen

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吡哆醛 5'-磷酸 (PLP) 依赖性酶的机制需要底物形成共价“外部醛亚胺”中间体，该中间体强烈吸收 410 至 430 nm 之间的光。天冬氨酸转氨酶 (AAT) 是一种典型的 PLP 依赖性酶，可催化天冬氨酸和 α-酮戊二酸与草乙酸和谷氨酸的可逆相互转化。根据动力学同位素效应研究，已知天冬氨酸外部醛亚胺 C(α)-H 键去质子化生成碳阴离子醌类中间体在热 AAT 反应中部分限制速率。基于溶液中席夫碱的研究，我们发现 430 nm 外部醛亚胺吸收带的激发增加了 AAT 的稳态催化活性，这归因于 C(α)-H 去质子化的光增强。蓝光 (250 mW) 照明观察到 WT AAT 活性增强 2.3 倍，无活性 K258A 突变体增强 530 倍，水中 PLP-Asp 希夫碱增强 58600 倍。这些不同程度的增强与不同环境中 C(α)-H 键的固有反应性相关，反应性较低的席夫碱表现出更大的增强。时间分辨光谱（范围从飞秒到分钟）用于研究水中和与 AAT 结合的 PLP 氨基酸席夫碱中 C(α)-H 键断裂的光活化性质。与热途径不同，光活化途径涉及三重态，其 C(α)-H pK(a) 估计比水中 PLP-Val 希夫碱的基态低 11 至 19 个单位。

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Light-Enhanced Catalysis by Pyridoxal Phosphate-Dependent Aspartate Aminotransferase

The mechanisms of pyridoxal 5'-phosphate (PLP)-dependent enzymes require substrates to form covalent "external aldimine" intermediates, which absorb light strongly between 410 and 430 nm. Aspartate aminotransferase (AAT) is a prototypical PLP-dependent enzyme that catalyzes the reversible interconversion of aspartate and α-ketoglutarate with oxalacetate and glutamate. From kinetic isotope effects studies, it is known that deprotonation of the aspartate external aldimine C(α)-H bond to give a carbanionic quinonoid intermediate is partially rate limiting in the thermal AAT reaction. We show that excitation of the 430-nm external aldimine absorption band increases the steady-state catalytic activity of AAT, which is attributed to the photoenhancement of C(α)-H deprotonation on the basis of studies with Schiff bases in solution. Blue light (250 mW) illumination gives an observed 2.3-fold rate enhancement for WT AAT activity, a 530-fold enhancement for the inactive K258A mutant, and a 58600-fold enhancement for the PLP-Asp Schiff base in water. These different levels of enhancement correlate with the intrinsic reactivities of the C(α)-H bond in the different environments, with the less reactive Schiff bases exhibiting greater enhancement. Time-resolved spectroscopy, ranging from femtoseconds to minutes, was used to investigate the nature of the photoactivation of C(α)-H bond cleavage in PLP-amino acid Schiff bases both in water and bound to AAT. Unlike the thermal pathway, the photoactivation pathway involves a triplet state with a C(α)-H pK(a) that is estimated to be between 11 and 19 units lower than the ground state for the PLP-Val Schiff base in water.

更新日期：2010-12-01

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