Chan-Huot, Monique and Dos, Alexandra and Zander, Reinhard and Sharif, Shasad and Tolstoy, Peter M. and Compton, Shara and Fogle, Emily and Toney, Michael D. and Shenderovich, Ilya and Denisov, Gleb S. and Limbach, Hans-Heinrich (2013) 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. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 135 (48). pp. 18160-18175. ISSN 0002-7863,
Full text not available from this repository. (Request a copy)Abstract
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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | NUCLEAR-MAGNETIC-RESONANCE; COLI TRYPTOPHAN SYNTHASE; TYROSINE PHENOL-LYASE; SOLID-STATE; ESCHERICHIA-COLI; ACTIVE-SITE; CRYSTAL-STRUCTURES; SCHIFF-BASES; CARBINOLAMINE FORMATION; SECONDARY STRUCTURE; |
| Subjects: | 500 Science > 540 Chemistry & allied sciences |
| Divisions: | Chemistry and Pharmacy > Central Analytical Services |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 24 Mar 2020 06:58 |
| Last Modified: | 24 Mar 2020 06:58 |
| URI: | https://pred.uni-regensburg.de/id/eprint/15540 |
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