NMR Studies of Protonation and Hydrogen Bond States of Internal Aldimines of Pyridoxal 5′-Phosphate Acid-Base in Alanine Racemase, Aspartate Aminotransferase, and Poly-L-lysine
NMR Studies of Protonation and Hydrogen Bond States of Internal Aldimines of Pyridoxal 5′-Phosphate Acid-Base in Alanine Racemase, Aspartate Aminotransferase, and Poly-L-lysine
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DOI：
10.1021/ja408988z
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发表时间：
2013-12-04
影响因子：
15
通讯作者：
Limbach, Hans-Heinrich
中科院分区：
文献类型：
--
作者：
Chan-Huot, Monique;Dos, Alexandra;Limbach, Hans-Heinrich
利用N-15固体核磁共振技术，我们研究了丙氨酸消旋酶（AlaR）、天冬氨酸转氨酶（AspAT）和聚-L-赖氨酸中作为内部醛亚胺连接的辅因子吡哆醛5 '-磷酸（PLP）的质子化和氢键状态。PLP的吡啶氮的质子化和从酚氧（烯醇亚胺形式）到醛亚胺氮（酮烯胺形式）的偶联质子转移通常被认为是酶催化反应的初始步骤（transimination）的先决条件。实际上，使用N-15 NMR和AspAT中的H-键相关性，我们观察到强的酒石酸-吡啶氮H-键，其中H位于氮上。在水合作用之后，这种氢键得以保持。相比之下，在固体冻干AlaR的情况下，我们发现吡啶氮既不质子化也不氢键合到近端精氨酸侧链。然而，水合作用与吡啶形成弱氢键。为了阐明AlaR是如何被激活的，我们对用聚-L-赖氨酸冻干形成的同位素标记的PLP醛亚胺进行了C-13和N-15固态NMR实验。在干燥固体中，仅观察到烯醇亚胺互变异构体。然而，一个快速可逆的质子转移涉及酮烯胺互变异构体后，观察到处理与气态水或气态干燥HCl。水解需要水和HCl的作用。在pH 9下，天冬氨酸与外部醛亚胺的形成也产生通过与第二个天冬氨酸的相互作用而稳定的酮烯胺形式，可能是通过与酚氧的H-键。我们假设，O-质子化是一个有效的机制，为PLP的激活，是N-质子化，是不能N-质子化的酶采用这种机制。
Using N-15 solid-state NMR, we have studied protonation and H-bonded states of the cofactor pyridoxal 5'-phosphate (PLP) linked as an internal aldimine in alanine racemase (AlaR), aspartate aminotransferase (AspAT), and poly-L-lysine. Protonation of the pyridine nitrogen of PLP and the coupled proton transfer from the phenolic oxygen (enolimine form) to the aldimine nitrogen (ketoenamine form) is often considered to be a prerequisite to the initial step (transimination) of the enzyme-catalyzed reaction. Indeed, using N-15 NMR and H-bond correlations in AspAT, we observe a strong aspartate-pyridine nitrogen H-bond with H located on nitrogen. After hydration, this hydrogen bond is maintained. By contrast, in the case of solid lyophilized AlaR, we find that the pyridine nitrogen is neither protonated nor hydrogen bonded to the proximal arginine side chain. However, hydration establishes a weak hydrogen bond to pyridine. To clarify how AlaR is activated, we performed C-13 and N-15 solid-state NMR experiments on isotopically labeled PLP aldimines formed by lyophilization with poly-L-lysine. In the dry solid, only the enolimine tautomer is observed. However, a fast reversible proton transfer involving the ketoenamine tautomer is observed after treatment with either gaseous water or gaseous dry HCl. Hydrolysis requires the action of both water and HCl. The formation of an external aldimine with aspartic acid at pH 9 also produces the ketoenamine form stabilized by interaction with a second aspartic acid, probably via a H-bond to the phenolic oxygen. We postulate that O-protonation is an effectual mechanism for the activation of PLP, as is N-protonation, and that enzymes that are incapable of N-protonation employ this mechanism.