8-氨基-7-氧代壬酸合酶的机制：光谱，动力学和晶体学研究。,Biochemistry

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8-氨基-7-氧代壬酸合酶的机制：光谱，动力学和晶体学研究。
Biochemistry ( IF 2.9 ) Pub Date : 2000-01-21 , DOI: 10.1021/bi991620j
S P Webster ₁ , D Alexeev , D J Campopiano , R M Watt , M Alexeeva , L Sawyer , R L Baxter

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8-Amino-7-oxononanoate合酶（也称为7-keto-8-氨基壬酸合酶，EC 2.3.1.47）是吡咯醛5'-磷酸盐依赖性酶，可催化L-丙氨酸与庚二酰-CoA的脱羧缩合。以立体定向的方式形成8（S）-氨基-7-氧代壬酸酯。这是生物素生物合成的第一步。大肠杆菌AONS的机理已通过光谱，动力学和晶体学技术进行了研究。完整酶的X射线结构已以1.7 A的分辨率精制（R = 18.6％，R（游离）=21。2％），显示内部醛亚胺的亚胺键平面偏离吡啶飞机。酶-产品外部亚胺亚胺复合物的结构已以2.0 A的分辨率精制（R = 21.2％，R（free）= 27。8％），并显示吡啶环相对于内部醛亚胺的旋转，以及C端结构域的显着构象变化和活性位点氢键的细微重排。反应的第一步是L-丙氨酸外部醛亚胺的形成，反应迅速（k（1）= 2 x 10（4）M（-）（1）s（-）（1））。用D-丙氨酸（不是底物）形成外部醛亚胺的速度明显较慢（k（1）= 125 M（-）（1）s（-）（1））。在庚二酰辅酶A存在下，D-丙氨酸与AONS的结合增强了约2倍。仅在向预先形成的L-丙氨酸外部醛亚胺络合物中添加庚二酰-CoA时，才发生显着的底物醌类形成，并且其之前存在明显的滞后相（约30 ms），这表明庚二酰-CoA的结合会导致酶的构象转变外部醛亚胺络合物。这种转变是通过建模推断的，要求围绕外部醛亚胺络合物的Calpha-N键进行旋转，从而促进了Lys236对Calpha质子的提取。这些结果已合并形成AONS催化的详细机理途径，该途径可应用于α-氧胺合酶亚家族的其他成员。促进Lys236对Calpha质子的抽象。这些结果已合并形成AONS催化的详细机理途径，该途径可应用于α-氧胺合酶亚家族的其他成员。促进Lys236对Calpha质子的抽象。这些结果已合并形成AONS催化的详细机理途径，该途径可应用于α-氧胺合酶亚家族的其他成员。

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Mechanism of 8-amino-7-oxononanoate synthase: spectroscopic, kinetic, and crystallographic studies.

8-Amino-7-oxononanoate synthase (also known as 7-keto-8-aminopelargonate synthase, EC 2.3.1.47) is a pyridoxal 5'-phosphate-dependent enzyme which catalyzes the decarboxylative condensation of L-alanine with pimeloyl-CoA in a stereospecific manner to form 8(S)-amino-7-oxononanoate. This is the first committed step in biotin biosynthesis. The mechanism of Escherichia coli AONS has been investigated by spectroscopic, kinetic, and crystallographic techniques. The X-ray structure of the holoenzyme has been refined at a resolution of 1.7 A (R = 18.6%, R(free) = 21. 2%) and shows that the plane of the imine bond of the internal aldimine deviates from the pyridine plane. The structure of the enzyme-product external aldimine complex has been refined at a resolution of 2.0 A (R = 21.2%, R(free) = 27.8%) and shows a rotation of the pyridine ring with respect to that in the internal aldimine, together with a significant conformational change of the C-terminal domain and subtle rearrangement of the active site hydrogen bonding. The first step in the reaction, L-alanine external aldimine formation, is rapid (k(1) = 2 x 10(4) M(-)(1) s(-)(1)). Formation of an external aldimine with D-alanine, which is not a substrate, is significantly slower (k(1) = 125 M(-)(1) s(-)(1)). Binding of D-alanine to AONS is enhanced approximately 2-fold in the presence of pimeloyl-CoA. Significant substrate quinonoid formation only occurs upon addition of pimeloyl-CoA to the preformed L-alanine external aldimine complex and is preceded by a distinct lag phase ( approximately 30 ms) which suggests that binding of the pimeloyl-CoA causes a conformational transition of the enzyme external aldimine complex. This transition, which is inferred by modeling to require a rotation around the Calpha-N bond of the external aldimine complex, promotes abstraction of the Calpha proton by Lys236. These results have been combined to form a detailed mechanistic pathway for AONS catalysis which may be applied to the other members of the alpha-oxoamine synthase subfamily.

更新日期：2019-11-01

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