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

Abstract

Using (15)N 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 (15)N 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 (13)C and (15)N 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.

Influence of aqueous dimethyl sulfoxide on pyridoxine protonation and tautomerization

“…For example, the influence of pyridine nitrogen protonation of more complex compound, PLP, on PLP or its derivatives reactions (e.g. transamination) is noticeable [5][6][7][8]. However, the effect of pyridoxine heteronitrogen protonation on PN activity in biochemical reactions is still unknown.…”

Solution‐State 17O Quadrupole Central‐Transition NMR Spectroscopy in the Active Site of Tryptophan Synthase

“…[1] Delineating the factors that fine-tune PLP for a particular reaction remains of great interest. [1–2] Studies of PLP in model compounds have demonstrated the important roles that protonation states and tautomerization play in directing reaction specificity. [3] …”

“…Tracking the equilibrium involved in tautomerization can be accomplished by interrogating the heteronuclei involved in the proton exchange, yet to date this has largely been limited to 15 N chemical shift measurements. [2, 4] Oxygen, the other key atomic species often involved in tautomerism, is not yet a standard nuclear probe in biological NMR spectroscopy, despite the potential wealth of chemical information it can provide. Here we report the first application of 17 O quadrupole central transition (QCT) NMR to interrogate kinetically-competent species under conditions of active catalysis, probing two quasi-stable intermediates in the catalytic cycle of the 143 kDa, PLP-dependent enzyme tryptophan synthase (TS).…”

A subfamily of PLP-dependent enzymes specialized in handling terminal amines

“…Hydrogen bonding the phenolic oxygen may be important for modulating the pK a of the internal aldimine (the Schiff base of the PLP with the catalytic lysine) [66] and also the reactivity of PLP [67].…”