Pyridoxal 5 0 -phoshate Schiff base in Citrobacter freundii tyrosine phenol-lyase Ionic and tautomeric equilibria Natalia P. Bazhulina, Yurii V. Morozov, Anastasia I. Papisova and Tatyana V. Demidkina Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia Spectral properties of the internal Schiff base in tyrosine phenol-lyase have been investigated in the presence of an activating cation K 1 and a cation-inhibitor Na 1 . The holoenzyme absorption spectra in the pH range 6.5±8.7 were recorded in the presence of K 1 . No apparent pK a 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 0 -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 1 with Na 1 . This replacement leads to a significant decrease in the pK a value of pyridinium nitrogen of the pyridoxal-P . Keywords: tyrosine phenol-lyase; internal Schiff base; monovalent cations; ionic forms; tautomeric forms. Bacterial tyrosine phenol-lyase (EC 4.1.99.2) is a pyridoxal- P-dependent enzyme that catalyses b-elimination reaction of l-tyrosine to give phenol, pyruvic acid, and ammonia (see below). The enzyme is found mainly in the Enterobacteriaceae family [1]. The enzyme from Citrobacter freundii is the best studied. The tyrosine phenol-lyase gene from this bacterium was cloned and the protein amino-acid sequence was deduced from cDNA structure. The molecule of the enzyme consists of four identical polypeptide chains of 51.4 kDa each [2]. X-ray studies of the C. freundii apoenzyme [2] and C. freundii and Erwinia herbicola holoenzymes [3,4] have shown that the tetramer consists of two catalytical dimers, each of which contains an active site formed by the residues of two subunits. In each subunit, the :-amino group of lysine residue (Lys257) forms an aldimine bond (the internal Schiff base, the internal aldimine) with the coenzyme aldehyde group. Tyrosine phenol-lyase requires monovalent cations for its activity. Most monovalent cations are enzyme activators, whereas Na 1 is inhibitor [5]. The monovalent cation- activator (one in each subunit) is bound on the border between the catalytic dimer subunits with a co-ordination number of 7 [3,4]. The first step of various reactions of amino-acid metabolism catalysed by pyridoxal-P-dependent enzymes is the formation of the external Schiff base (the external aldimine) as a result of transaldimination between the internal aldimine and the amino- acid substrate. Knowledge of ionic and tautomeric forms of the internal Schiff base is necessary for understanding of the mechanism of transaldimination step. To achieve this, we have studied spectroscopic properties of the internal aldimine of recombinant tyrosine phenol-lyase from C. freundii in the presence of K 1 and Na 1 cations. MATERIALS AND METHODS Materials Lactate dehydrogenase from rabbit muscle, pyridoxal-P , NADH, l-tyrosine, dithiothreitol, d,l-penicillamine, KCl and NaCl were purchased from Sigma. Other reagents were from Reachim (Russia) of the puriss grade. All buffer solutions contained 2  10 24 m dithiothreitol and were prepared with deionized water. Eur. J. Biochem. 267, 1830±1836 (2000) q FEBS 2000 Correspondence to T. V. Demidkina, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, 117984 Moscow, Russia, Fax: 1 7095 135 14 05, Tel.: 1 7095 135 98 58, E-mail: tvd@genome.eimb.relarn.ru Enzymes: tyrosine phenol-lyase (EC 4.1.99.2), tryptophan indole-lyase (EC 4.1.99.1); glutamate decarboxylase (EC 4.1.1.15); aspartate aminotransferase (EC 2.6.1.1); aromatic amino-acid aminotransferase (EC 2.6.1.57); l-lactic dehydrogenase (EC 1.1.1. 27). (Received 27 October 1999, revised 26 January 2000, accepted 27 January 2000)