Pyridoxal 5'-phoshate schiff base in Citrobacter freundii tyrosinephenol-lyase. Ionic and tautomeric equilibria.

Spectral properties of the internal Schiff base in tyrosine phenol-lyase have been investigated in the presence of an activating cation K+ and a cation-inhibitor Na+. The holoenzyme absorption spectra in the pH range 6.5-8.7 were recorded in the presence of K+. No apparent pKa value of the coenzyme chromophore was found in this pH range, indicating that the internal Schiff base does not change its ionic form on going from pH 6.5 to 8.7. To determine the ionic state and tautomeric composition of the Schiff base in tyrosine phenol-lyase, the absorption and circular dichroism spectra were analyzed using lognormal distribution curves. The predominant form of the internal Schiff base is that with protonated pyridinium and aldimine nitrogen atoms and deprotonated 3'-hydroxy group, i.e. the ketoenamine. This form is in prototropic equilibrium with its enolimine tautomer. The internal aldimine ionic form is changed upon replacement of K+ with Na+. This replacement leads to a significant decrease in the pKa value of pyridinium nitrogen of the pyridoxal-P.