Substrate-Recognition Mechanism of Aromatic Amino Acid Aminotransferase

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Most Aminotransferases are active toward both neutral and acidic amino acids. This research aims to investigate the dual substrate-recognition mechanism of aminotransferases using aromatic amino acid aminotransferase (ArAT) as a model enzyme.1. The omega- and alpha- carboxylate groups of acidic amino acids are recognized by Arg292 and Arg386 of ArAT,respectively. The hydrogen-bonding network of Arg 386-Asn194-PLP was found to be critical for modulating the electronic status of PLP by substrate binding. This reflects that the alpha-carboxylate group is common to all amino acid substrates, and the activation of enzyme through substrate binding should involve the commom structural motif of amino acid substrates.2. Analysis of the reaction of beta-hydroxylated quasisubstrates and the wild-type and [Tyr70*Phe] mutant ArAT showed that the acidic and aromatic amino acids bind to the enzyme in almost identical conformations. This also indicated that the side chain of Arg292 must take altered conformations depending upon the nature of amino acid substrates, acidic or aromatic.3. Studies on the Arg292 mutant enzymes showed that the guanidinium group of Arg292 is important for the recognition of the aromatic ring of substrates. This indicated either that the Arg 292 side chain forms a part of the pocket that accepts the aromatic ring or that the guanidinium group required for the side chain of residue 292 to undergo the conformational change in order to create the pocket.4. In order to analyze further the mechanism of substrate recognition at an atomic level, X-ray crystallographic analysis of ArAT is required. Although the crystal of E.coli ArAT has not been obtained, Paracoccus ArAT was found to be crystallized well enough to be used for X-ray analysis. Structural analyzes of the enzyme itself and the complexes with substrate analogs are now under way.