| Research Abstract |
We have examined the functional role of some active site residues of E.coli aspartate, aromatic, and branched amino acid aminotransferases. Our findings are as follows :
1. artate aminotransferases (AspAT).
1) The negative charge on Asp222 stabilyzes the positive charge on N (1) of the coenzyme, pyridoxal phosphate, and aids the abstraction of an alpha-proton from the substrate during the catalysis. Asp222 is also important for supporting the coenzyme ring in position.
2) Asn194 functions in substrate binding through hydrogen bonding to the substrate carboxylate and/or holding the side chain of Arg386, which interacts with the substrate alpha-carboxylate, in a position suitable for substrate binding. Further, we propose that the idea that Asn194 lowers the pKa of the imine nitrogen of the internal aldimine bond to facilitate a transaldimination step.
3) The X-ray crystallography revealed the conformational change upon binding of the substrate (inhibitor) to close the active site. At the sa
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me time, neutralization of the positive charge on these two arginine residues increases the pKa of the internal aldimine through direct Coulombic interaction and Arg386-Asn194-coenzyme hydrogen bonding network, facilitating transaldimination step.
2. Aromatic amino acid aminotransferase (arAT).
ArAT from E.coli was overexpressed in E.coli cells, purified, and characterized. ArAT and AspAT showed overlapping substrate specificity. Both of the enzymes were active toward dicarboxylic substrates. However, ArAT showed 10^3-fold higher activity toward aromatic substrates than AspAT,and this was in part ascribed to the active site hydrophobicity. Asn194 and Arg386 of ArAT had similar effect on the pKa of aldimine as observed for AspAT.
3. Branched-amino acid aminotransferase (BrAT).
Preliminary X-ray characterization of BrAT was achieved. BrAT catalyzes pro-R C-4' hydrogen transfer through the coenzyme-substrate Schiff base intermediate as observed in D-amino acid aminotransferase, in contrast to other various aminotransferases catalysing the pro-S hydrogen transfer. Less
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