¹⁵N Nuclear Magnetic Resonance Studies of Acid−Base Properties of Pyridoxal-5‘-Phosphate Aldimines in Aqueous Solution

Abstract

By use of 15N NMR spectroscopy, we have measured the pKa values of the aldimines 15N-(pyridoxyl-5'-phosphate-idine)-methylamine (2a), N-(pyridoxyl-5'-phosphate-15N-idine)-methylamine (2b), and 15N-(pyridoxyl-idine)-methylamine (3). These aldimines model the cofactor pyridoxal-5'-phosphate (PLP, 1) in a variety of PLP-dependent enzymes. The acid-base properties of the aldimines differ substantially from those of the free cofactor in the aldehyde form 1a or in the hydrated form 1b, which were also investigated using 15N NMR for comparison. All compounds contain three protonation sites, the pyridine ring, the phenol group, and the side chain phosphate (1, 2) or hydroxyl group (3). In agreement with the literature, 1a exhibits one of several pKas at 2.9 and 1b at 4.2. The 15N chemical shifts indicate that the corresponding deprotonation occurs partially in the pyridine and partially in the phenolic site, which compete for the remaining proton. The equilibrium constant of this ring-phenolate tautomerism was measured to be 0.40 for 1a and 0.06 for 1b. The tautomerism is essentially unaltered above pH 6.1, where the phosphate group is deprotonated to the dianion. This means that the pyridine ring is more basic than the phenolate group. Pyridine nitrogen deprotonation occurs at 8.2 for 1a and at 8.7 for 1b. By contrast, above pH 4 the phosphate site of 2 is deprotonated, while the pyridine ring pKa is 5.8. The Schiff base nitrogen does not deprotonate below pH 11.4. When the phosphate group is removed, the pKa of the Schiff base nitrogen decreases to 10.5. The phenol site cannot compete for the proton of the Schiff base nitrogen and is present in the entire pH range as a phenolate, preferentially hydrogen bonded to the solvent. The intrinsic 15N chemical shifts provide information about the hydrogen bond structures of the protonated and unprotonated species involved. Evidence is presented that the intramolecular OHN hydrogen bond of PLP aldimines is broken in aqueous solution. The coupling between the inter- and intramolecular OHN hydrogen bonds is also lost in this environment. The pyridine ring of the PLP aldimines is not protonated in aqueous solution near neutral pH. The basicity of the aldimine nitrogens would be even lower without the doubly negatively charged phosphate group. Protonation of both the Schiff base and pyridine nitrogens has been discussed as a prerequisite for catalytic activity, and the implications of the present findings for PLP catalysis are discussed.

Molecular dynamics simulations of the intramolecular proton transfer and carbanion stabilization in the pyridoxal 5′-phosphate dependent enzymes l-dopa decarboxylase and alanine racemase

“…The pyridine nitrogen of the external aldimine substrate has a pKa of about 5.8 in aqueous solution [1,12]. However, in the active sites of most PLP-dependent enzymes, the pyridine nitrogen is assumed to be protonated due to stabilization through ion-pair interactions with a basic residue such as Glu and Asp or hydrogen bonding to polar residues such as Ser and Thr.…”

“…A protonated pyridine strongly favors the N-protonated Schiff base in the solid state and in polar aprotic solvents. However, the intramolecular hydrogen bond is not coupled to the protonation state of the pyridine ring in aqueous solution [12]. This has been attributed to competing hydrogen bonding interactions with water, causing the imino group to rotate out of the aromatic plane.…”

“…Toney and Limbach and their coworkers demonstrated that the intramolecular proton transfer is coupled to the protonation state of the pyridine ring, which favors the N-protonated Schiff base (PSB) in solid state and in polar aprotic solvent when the pyridine ring is protonated [9,10]. However, there is no coupling in aqueous solution due to competition between intramolecular and intermolecular hydrogen bonds [11,12]. …”

“…The external aldimine between PLP and an amino acid substrate consists of three titratable sites in addition to the phosphate group, [10,13] including the pyridine nitrogen, the 3-oxo oxygen and the imine nitrogen of the Schiff base linkage; the specific protonation state and site are critical to catalysis [12]. Under physiological conditions, the external aldimine would retain only one proton, whereas the protonation of the pyridine nitrogen site is strongly dependent on the amino acid with which it forms a specific hydrogen bond in the active site.…”

Aspartate aminotransferase: An old dog teaches new tricks

“…[52] In crystalline samples of both the unliganded and maleate-liganded enzyme forms at low pH, the pyridine nitrogen is fully protonated. The Asp222 oxygen - pyridine nitrogen distance is 2.63 Å in the unliganded crystals and somewhat shorter at 2.60 Å in the closed, maleate-liganded conformation, in agreement with X-ray structures.…”