The substrate activation process in the catalytic reaction of Escherichia coli aromatic amino acid aminotransferase

Aromatic amino acid aminotransferase is active toward both aromatic and dic arboxylic amino acids, and the mechanism for this dual substrate recognitio n 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 Sch iff base formed between the coenzyme pyridoxal 5'-phosphate and Lys258 or t he substrate increases successively from 6.6 in the unliganded enzyme to si milar to8.8 in the Michaelis complex and to >10.5 in the external Schiff ba se complex. Mutations of Arg292 and Arg386 to Leu, which mimic neutralizati on of the positive charges of the two arginine residues by the ligand carbo xylate groups, increased the Schiff base pK(a) by 0.1 and 0.7 unit, respect ively. In contrast to these moderate effects of the Arg mutations, the clea vage of the Lys258 side chain of the Schiff base, which was brought about b y preparing a mutant enzyme in which Lys258 was changed to Ala and the Schi ff 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 enzym e. The observation indicates that the Schiff base pK(a) in the enzyme is lo wered 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 t he 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 e lectrostatic contribution from the omega -carboxylate group of the substrat e, 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 be en demonstrated in pyridoxal enzymes other than aspartate aminotransferase, and suggests the generality of the mechanism in the catalysis of aminotran sferases related to aspartate aminotransferase.