The Novel Substrate Recognition Mechanism Utilized by Aspartate Aminotransferase of the Extreme Thermophile Thermus thermophilus HB8

• DOI: 10.1074/jbc.273.45.29554
• Publication Date: 2002-07-26T15:02:22Z
• Authors: Yuko Nobe; Shin-ichi Kawaguchi; Hideaki Ura; Tadashi Nakai; Ken Hirotsu; Ryuichi Kato; Seiki Kuramitsu
• Keywords: Thermus thermophilus; Thermus

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