The six mutations, referred to as the Hex mutations, that together have been shown to convert Escherichia coli aspartate aminotransferase (AATase) specificity to be substantially like that of E. coli tyrosine aminotransferase (TATase) are dissected into two groups, (T109S/N297S) and (V39L/K41Y/T47I/N69L). The letters on the left and right of the numbers designate AATase and TATase residues, respectively. The T109S/N297S pair has been investigated previously. The latter group, the "Grease" set, is now placed in the AATase framework, and the retroGrease set (L39V/Y41K/I47T/L69N) is substituted into TATase. The Grease mutations in the AATase framework were found primarily to lower K M s for both aromatic and dicarboxylic substrates. In contrast, retroGrease TATase exhibits lowered k cat s for both substrates. The six retroHex mutations, combining retroGrease and S109T/S297N, were found to invert the substrate specificity of TATase, creating an enzyme with a nearly ninefold preference (k cat /K M ) for aspartate over phenylalanine. The retroHex mutations perturb the electrostatic environment of the pyridoxal phosphate cofactor, as evidenced by a spectrophotometric titration of the internal aldimine, which uniquely shows two pK a s, 6.1 and 9.1. RetroHex was also found to have impaired dimer stability, with a K D for dimer dissociation of 350 nM compared with the wild type K D of 4 nM. Context dependence and additivity analyses demonstrate the importance of interactions of the Grease residues with the surrounding protein framework in both the AATase and TATase contexts, and with residues 109 and 297 in particular. Context dependence and cooperativity are particularly evident in the effects of mutations on k cat /K M (Asp). Effects on k cat /K M (Phe) are more nearly additive and context independent.Keywords: Aminotransferase; chimera; context dependence; protein/genetic engineering; pyridoxal phosphate; substrate specificity A central problem in the area of structural biology is that of identifying the functionally important amino acid residues of an enzyme and of quantitating their individual and context dependent contributions. Many of the proteins whose sequences have been elucidated by genomics remain uncharacterized. Families of such proteins, which may be quite diverse in function or substrate specificity, provide an opportunity to study putative functionally important residues by creating chimeras in which residues from one sequence are transferred into another. The results have implications both for the study of protein function and for enzyme redesign.Several successful examples of enzyme substrate specificity redesign inspired by analyses of homologous sequences have been reported. Subtilisin has been engineered 1 Present address: