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 PROBETHE ENZYME-SUBSTRATE INTERACTIONS/

Escherichia coli aromatic amino acid aminotransferase (ArAT) catalyzes transamination reactions of both dicarboxylic amino acids and aromati c amino acids. Because both reactions are supposed to occur in a singl e reaction center, whether ArAT provides alternative binding sites for the two different types of substrate side chains has been an intrigui ng question. This was probed by spectroscopic analysis of the complexe s of beta-hydroxylated substrates and the wild-type and [Tyr70 --> Phe ] mutant enzymes. Both L-elythro-3-hydroxyaspartate and L-erythro-3-ph enylserine reacted with the wild-type ArAT to give an absorption maxim um at around 500 nm, reflecting the formation of the quinonoid interme diate. When the hydroxy group of Tyr70 of ArAT was deleted by replacem ent of the residue with phenylalanine, the 500-nm absorption greatly d ecreased in either of the ArAT-beta-hydroxy amino acid complexes, show ing 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 quas isubstrates are essentially identical in their enzyme-bound forms. Thi s implies that the phenyl group of the substrate phenylalanine occupie s the same region as that occupied by the beta-carboxyl group of the s ubstrate aspartate, and the region should be near Arg292, the residue that binds the beta-carboxylate group of substrates. The [Arg292 --> A la] or [Arg292 --> Leu] mutation increased the K-m values for aromatic amino acids 5-10-fold, and the [Arg292 --> Lys] mutation increased th ese values 10-100-fold, without affecting the k(cat) values. This show s that the side chain of Arg292 is partially involved in the binding o f the aromatic ring of substrates to ArAT.