Aspartate aminotransferase (AspAT) is a unique enzyme that can react with two types of substrate with quite different properties, acidic substrates, such as aspartate and glutamate, and neutral substrates, although the catalytic group Lys-258 acts on both types of substrate. The dynamic properties of the substrate-binding site are indispensable to the interaction with hydrophobic substrates (Kawaguchi, S., Nobe, Y., Yasuoka, J., Wakamiya, T., Kusumoto, S., and Kuramitsu, S. (1997) J. Biochem. (Tokyo) 122, 55-63). AspATs from various organisms are classified into two subgroups, Ia and Ib. The former includes AspATs from Escherichia coli and higher eukaryotes, whereas the latter includes those from Thermus thermophilus and many prokaryotes. The AspATs belonging to subgroup Ia each have an Arg-292 residue, which interacts with the distal carboxyl groups of dicarboxylic (acidic) substrates, but the functionally similar residue of subgroup Ib AspATs has not been identified. In view of the x-ray crystallographic structure of T. thermophilus AspAT, we expected Lys-109 to be this residue in the subgroup Ib AspATs and constructed K109V and K109S mutants. Replacing Lys-109 with Val or Ser resulted in loss of activity toward acidic substrates but increased that toward the neutral substrate, alanine, considerably. These results indicate that Lys-109 is a major determinant of the acidic substrate specificity of subgroup Ib AspATs. Kinetic analysis of the interactions with neutral substrates indicated that T. thermophilus AspAT is subject to less steric hindrance and its substrate-binding pocket has a more flexible conformation than E. coli AspAT. A flexible active site in the rigid T. thermophilus AspAT molecule may explain its high activity even at room temperature.