| Research Abstract |
Aspartate aminotransferase (AspAT) has the essential cofactor, pyridoxal 5'-phosphate (a vitamin B_6 derivative) and catalyzes the reversible amino group transfer between L-aspartate ad 2-oxoglutarate. The system for studying the catalytic mechanism of AspAT was constructed. The nucleotide sequence of the gene for Escherichia coli AspAT was determined. The amino acid residues in the active site were replaced by site-directed mutagenesis. The wild-type and mutant AspATs were overproduced in the E. coli strain (TY103) lacking the gene for AspAT. The three-dimensional structures of the wild-type and mutant AspATs were determined.
A stopped-flow method monitoring the absorption change of the coenzyme clarified that the catalytic process (half-transamination reaction) of AspAT consists of the two substrate-binding steps in rapid equilibrium and an intramolecular rate-determining step.
In the rate-determining step, Lys258, Tyr70, Try225, and Asp222, which exist around the bound coenzyme, played important roles.
In the substrate binding, four interesting phenomena were observed.
(1) Arg is essential for recognizing the carboxyl group of the substrate. Lys cannot substitute for Arg.
(2) When Arg292 was replaced by Val or Leu, the substrate specificity of AspAT was changed from acidic to neutral substrates.
(3) The active site of AspAT is hydrophobic.
(4) AspAT has two pockets for accomomdating the side chain of a substrate, one for acidic substrate (Asp, Glu), and the other for hydrophobic substrate.
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