NMR Studies of Protonation and Hydrogen Bond States of Internal Aldimines of Pyridoxal 5鈥?Phosphate Acid鈥揃ase in Alanine Racemase, Aspartate Aminotransferase, and Poly-l

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• 作者：Monique Chan-Huot ; Alexandra Dos ; Reinhard Zander ; Shasad Sharif ; Peter M. Tolstoy ; Shara Compton ; Emily Fogle ; Michael D. Toney ; Ilya Shenderovich ; Gleb S. Denisov ; Hans-Heinrich Limbach
• 刊名：Journal of the American Chemical Society
• 出版年：2013
• 出版时间：December 4, 2013
• 年：2013
• 卷：135
• 期：48
• 页码：18160-18175
• 全文大小：716K
• 年卷期：v.135,no.48(December 4, 2013)
• ISSN：1520-5126

文摘

Using ¹⁵N solid-state NMR, we have studied protonation and H-bonded states of the cofactor pyridoxal 5鈥?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 ¹⁵N 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 ¹³C and ¹⁵N 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.