NMR Studies of the Stability, Protonation States, and Tautomerism of 13C- and 15N-Labeled Aldimines of the Coenzyme Pyridoxal 5′-Phosphate in Water

We have measured the pH-dependent H-1, C-13, and N-15 NMR spectra of pyridoxal 5'-phosphate (C-13(2)-PLP) mixed with equal amounts of either doubly N-15-labeled diaminopropane, N-15(alpha)-labeled. L-lysine, or N-15(epsilon)-labeled L-lysine as model systems for various intermediates of the transimination reaction in PLP-dependent enzymes. At low pH, only the hydrate and aldehyde forms of PLP and the free protonated diamines are present. Above pH 4, the formation of single- and double-headed aldimines (Schiff bases) with the added diamines is observed, and their C-13 and N-15 NMR parameters have been characterized. For 1:1 mixtures the single-headed aldimines dominate. In a similar way, the NMR parameters of the geminal diamine formed with diaminopropane at high pH are measured. However, no geminal diamine is formed with L-lysine. In contrast to the aldimine formed with the epsilon-amino group of lysine, the aldimine formed with the alpha-amino group is unstable at moderately high pH but dominates slightly below pH 10. By analyzing the NMR data, both the mole fractions of the different PLP species and up to 6 different protonation states including their pK(a) values were obtained. Furthermore, the data show that all Schiff bases are subject to a proton tautomerism along the intramolecular OHN hydrogen bond, where the zwitterionic form is favored before deprotonation occurs at high pH. This observation, as well as the observation that around pH 7 the different PLP species are present in comparable amounts, sheds new light on the mechanism of the transimination reaction.