The tyrosine (eTATase) and aspartate (eAATase) aminotransferases of Escherichia coli transaminate dicarboxylic amino acids with similar rate constants. However, eTATase exhibits -102-104-fold higher second-order rate constants for the transamination of aromatic amino acids than does eAATase. A series of natural and unnatural amino acid substrates was used to quantitate specificity differences for these two highly related enzymes. A general trend toward lower transamination activity with increasing side-chain length (extending from aspartate to glutamate to a-aminoadipate) is observed for both enzymes. This result suggests that dicarboxylate ligands associate with the two highly related enzymes in a similar manner. The high reactivity of the enzymes with L -A s~ and L-GIu can be attributed to an ion pair interaction between the side-chain carboxylate of the amino acid substrate and the guanidin0 group of the active site residue Arg 292 that is common to both enzymes. A strong linear correlation between side-chain hydrophobicity and transamination rate constants obtains for n-alkyl side-chain amino acid substrates with eTATase, but not for eAATase. The present kinetic data support a model in which eAATase contains one binding mode for all classes of substrate, whereas the active site of eTATase allows an additional mode that has increased affinity for hydrophobic amino acids.Keywords: aspartate aminotransferase; kinetics; substrate specificity; tyrosine aminotransferase Escherichia coli aspartate aminotransferase (eAATase; EC 2.6.1.1) and tyrosine aminotransferase (eTATase; EC 2.6.1.5) are PLP-containing transaminases that catalyze the reversible interconversion of amino acids and their corresponding a-keto acids. The bound cofactor acts as a transient amino group carrier, shuttling between the pyridoxal phosphate and pyridoxamine phosphate forms. The transamination reaction is described by a ping-pong bi-bi mechanism (Equation lA,B): with the side-chain carboxylate group of aspartate or glutamate (Kirsch et al., 1984;Malashkevich et al., 1993). Mutational stud-
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