| Research Abstract |
Substrate Recogntion of Pyridoxal Enzymes
X-ray crystallographic analyzes of aspartate aminotransferase (AST), aromatic amino acid aminotransferase (ArT) and branched-chain amino acid aminotransferase (BCT), in the presence of substrate analogue, domonstrated the mode for their substrate recognition.
In AST and ArT,the alpha and distal carboxylate groups of a dicarboxylic substrate form bifurcated hydrogen bonds with Arg386 and Arg292, respectively. However, the alpha-carboxylate group recognition of BCT was different : OH of Tyr95 and two peptide NH groups of beta-turn comprising Gly256-Ala259.
ArT accomodates the acidic and aromatic side chains of substrates at the same pocket by rearranging the hydrogen bond networks caused by reorientation of the side chains of some active site residues. Random mutation of AST gene by DNA shuffling gave new AST with substrate specificity for branched-chain amino acid.
Catalytic mechanism of Pyridoxal Enzymes
We could separate kinetically the two routes
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for association of AST and asparate, the one begins with the unprotonated aldimine and monoanionic aspartate, and the other with the protonated aldimine and dianionic aspartate. The earlier proposals have not considered the latter.
Lys303 of aromatic amino acid decarboxylase (AADC) was found to be the residue forming the internal aldimine. Examining the side reaction of [Lys303 Ala] enzyme, we could indicate that the lysine residue is not essential for the decarboxylation step, but important for the product release.
The conformational change of the putative flexible region of AADC introduced by the substrate binding was shown to be important for the external aldmine formation, mainly by analyzing the fragmentary enzyme cleaved at the bond inside the flexible region.
Tryptophanase is catalytically activ in alkaline pH in spite of inactive aldamine structure of the internal aldimine to be the major species. We could reveal that the aldamine structure was converted to the active ketoenamine structure upon the substrate-binding. Less
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