title: Analysis of the substrate-recognition mode of aromatic amino acid aminotransferase by combined use of quasisubstrates and site-directed mutagenesis: systematic hydroxy-group addition/deletion studies to probe the enzyme-substrate interactions.
abstract: Escherichia coli aromatic amino acid aminotransferase (ArAT) catalyzes transamination reactions of both dicarboxylic amino acids and aromatic amino acids. Because both reactions are supposed to occur in a single reaction center, whether ArAT provides alternative binding sites for the two different types of substrate side chains has been an intriguing question. This was probed by spectroscopic analysis of the complexes of beta-hydroxylated substrates and the wild-type and [Tyr70-->Phe] mutant enzymes. Both L-erythro-3-hydroxyaspartate and L-erythro-3-phenylserine reacted with the wild-type ArAT to give an absorption maximum at around 500 nm, reflecting the formation of the quinonoid intermediate. When the hydroxy group of Tyr70 of ArAT was deleted by replacement of the residue with phenylalanine, the 500-nm absorption greatly decreased in either of the ArAT-beta-hydroxy amino acid complexes, showing the presence of specific interactions, which stabilize the 500-nm absorbing quinonoid intermediates, between the phenolic hydroxy group of Tyr70 and the beta-hydroxy groups of the two quasisubstrates. From these results, it was concluded that the conformations of the two quasisubstrates are essentially identical in their enzyme-bound forms. This implies that the phenyl group of the substrate phenylalanine occupies the same region as that occupied by the beta-carboxyl group of the substrate aspartate, and the region should be near Arg292, the residue that binds the beta-carboxylate group of substrates. The [Arg292-->Ala] or [Arg292-->Leu] mutation increased the Km values for aromatic amino acids 5-10 fold, and the [Arg292-->Lys] mutation increased these values 10-100-fold, without affecting the kcat values. This shows that the side chain of Arg292 is partially involved in the binding of the aromatic ring of substrates to ArAT.
authors.affiliation: Department of Biochemistry, Osaka Medical College, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Hayashi
authors.forename: K
authors.initials: K
authors.lastname: Inoue
authors.forename: H
authors.initials: H
authors.lastname: Mizuguchi
authors.forename: H
authors.initials: H
authors.lastname: Kagamiyama
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D000637:Transaminases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D008024:Ligands; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D000637:Transaminases; C019729:aromatic amino acid aminotransferase