The activity and reaction specificity of tyrosine phenol-lyase regulated by monovalent cations.

This work was aimed at studying the effect of monovalent inorganic cations (Li+, Na+, K+, Rb+, Cs+, NH+4) on the catalytic and spectral characteristics of tyrosine phenol-lyase from Citrobacter intermedius. These cations were shown to influence the proportion of the beta-elimination reaction rate to the rate of side transamination reaction. Most of the monovalent cations are non-competitive activators of the beta-elimination reaction; Li+ exerts no effect on the enzyme activity in this reaction; Na+ is an inhibitor of the beta-elimination reaction. The activation of tyrosine phenol-lyase by monovalent cations stems from the creation of an active holoenzyme form (lambda max 420 nm) due to conformational rearrangements of the protein molecule.

Tyrosine phenol-lyase from Citrobacter intermedius. Factors controlling substrate specificity.

L-Amino acids are competitive inhibitors of tyrosine phenol-lyase from Citrobacter intermedius. For non-branched amino acids the correlation exists between -RTlnKi and side-chain hydrophobicity. Aspartic and glutamic acids are anomalously potent inhibitors taking into account low hydrophobicity of their side chains. This suggests the presence of an electrophilic group in the active site which interacts with the terminal carboxylic group of aspartic or glutamic acids. Tyramine, beta-phenylethylamine and tryptamine do not display detectable inhibition. The esters and amides of aromatic L-amino acids, D-phenylalanine and D-tryptophan are competitive inhibitors. The enzymatic isotope exchange of the alpha-proton in 2H2O was observed only in the case of L-amino acids. For L-phenylalanine and L-tryptophan it was shown to proceed with complete retention of configuration. The substrate specificity of tyrosine phenol-lyase is controlled during the stage of phenol elimination. The OH group in the para position of the ring is necessary for this stage to proceed. The same stage is also sensitive to the steric parameters of the substituent in the ring which ensures the second factor of control. When all the requirements of substrate specificity are fulfilled (L-tyrosine, 3-fluoro-L-tyrosine), the 'key' phenol-elimination step is not the rate-limiting one, the reaction velocity being determined by the preceding alpha-proton abstraction.

Crystallization and crystal data on tyrosine phenol-lyase.

Crystals of the apoenzyme of tyrosine phenol-lyase (EC 4.1.99.2), a pyridoxal 5'-phosphate-dependent enzyme from Citrobacter intermedius, have been grown by vapor diffusion of an ammonium sulfate solution to a protein solution. The crystals belong to space group P2(1)2(1)2, with dimensions of a = 75.5 A, b = 138.4 A and c = 94.1 A and diffract up to 2.7 A resolution. The asymmetric unit contains one half of the enzyme tetrameric molecule. Two heavy-atom derivatives of the crystals have been obtained.

Transamination catalysed by tyrosine phenol-lyase from Citrobacter intermedius.

The interactions of tyrosine phenol-lyase with its substrates: L-tyrosine and L-serine, and the competitive inhibitors: L-alanine, L-phenylalanine, L-m-tyrosine, were studied. It was demonstrated that the enzyme catalyzed a half-transamination reaction between substrates or inhibitors and the protein-bound pyridoxal phosphate. The products of this side-reaction, pyridoxamine phosphate and the respective keto acids, were identified. The kinetic parameters were determined for beta-elimination of L-tyrosine and of L-serine, and for the transamination of L-serine and the inhibitors used. The transfer of the amino group to the coenzyme takes place in the direction from amino acid to pyridoxal phosphate, but not in the opposite direction, i.e. the transamination is irreversible.