| pubmed-article:11112527 | pubmed:abstractText | Aromatic amino acid aminotransferase is active toward both aromatic and dicarboxylic amino acids, and the mechanism for this dual substrate recognition has been an issue in the enzymology of this enzyme. Here we show that, in the reactions with aromatic and dicarboxylic ligands, the pK(a) of the Schiff base formed between the coenzyme pyridoxal 5'-phosphate and Lys258 or the substrate increases successively from 6.6 in the unliganded enzyme to approximately 8.8 in the Michaelis complex and to >10.5 in the external Schiff base complex. Mutations of Arg292 and Arg386 to Leu, which mimic neutralization of the positive charges of the two arginine residues by the ligand carboxylate groups, increased the Schiff base pK(a) by 0.1 and 0.7 unit, respectively. In contrast to these moderate effects of the Arg mutations, the cleavage of the Lys258 side chain of the Schiff base, which was brought about by preparing a mutant enzyme in which Lys258 was changed to Ala and the Schiff base was reconstituted with methylamine, produced the Schiff base pK(a) value of 10.2, that being 3.6 units higher than that of the wild-type enzyme. The observation indicates that the Schiff base pK(a) in the enzyme is lowered by the torsion around the C4-C4' axis of the Schiff base and suggests that the pK(a) is mainly controlled by changing the torsion angle during the course of catalysis. This mechanism, first observed for the reaction of aspartate aminotransferase with aspartate [Hayashi, H., Mizuguchi, H., and Kagamiyama, H. (1998) Biochemistry 37, 15076-15085], does not require the electrostatic contribution from the omega-carboxylate group of the substrate, and can explain why in aromatic amino acid aminotransferase the aromatic substrates can increase the Schiff base pK(a) during catalysis to the same extent as the dicarboxylic substrates. This is the first example in which the torsion pK(a) coupling of the pyridoxal 5'-phosphate Schiff base has been demonstrated in pyridoxal enzymes other than aspartate aminotransferase, and suggests the generality of the mechanism in the catalysis of aminotransferases related to aspartate aminotransferase. | lld:pubmed |
