THE NOVEL SUBSTRATE RECOGNITION MECHANISM UTILIZED BY ASPARTATE-AMINOTRANSFERASE OF THE EXTREME THERMOPHILE THERMUS-THERMOPHILUS HB8

Aspartate aminotransferase (AspAT) is a unique enzyme that can react w ith two types of substrate with quite different properties, acidic sub strates, such as aspartate and glutamate, and neutral substrates, alth ough 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. B iochem. (Tokyo) 122, 55-63). AspATs from various organisms are classif ied into two subgroups, Ia and Ib. The former includes AspATs from Esc herichia 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 fu nctionally similar residue of subgroup Ib AspATs has not been identifi ed. In view of the x-ray crystallographic structure of T. thermophilus AspAT, we expected Lys-109 to be this residue in the subgroup Ib AspA Ts and constructed K109V and K109S mutants. Replacing Lys-109 with Val or Ser resulted in loss of activity toward acidic substrates but incr eased that toward the neutral substrate, alanine, considerably. These results indicate that Lys-109 is a major determinant of the acidic sub strate specificity of subgroup Tb AspATs. Kinetic analysis of the inte ractions with neutral substrates indicated that T. thermophilus AspAT is subject to less steric hindrance and its substrate-binding pocket h as 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.