Site-directed mutagenesis of a hexapeptide segment involved in substrate recognition of phenylalanine dehydrogenase from Thermoactinomyces intermedius.
Phenylalanine dehydrogenase from Thermoactinomyces intermedius and leucine dehydrogenase from Bacillus stearothermophilus show a 59% sequence similarity in their substrate-binding domains, although their substrate specificities are different. We prepared a phenylalanine dehydrogenase mutant enzyme whose inherent hexapeptide segment (124F-V-H-A-A-129R) in the substrate-binding domain was replaced by the corresponding part of leucine dehydrogenase (M-D-I-I-Y-Q) in order to investigate the mechanism of substrate recognition by phenylalanine dehydrogenase. The catalytic efficiencies (kcat/Km) of the mutant enzyme with aliphatic amino acids and aliphatic keto acids as substrates were 0.5 to 2% of those of the wild-type enzyme. In contrast, the efficiencies for L-phenylalanine and phenylpyruvate decreased to 0.008 and 0.035% of those of the wild-type enzyme, respectively. These results suggest that the hexapeptide segment plays an important role in the substrate recognition by phenylalanine dehydrogenase.
Kataoka
K
K
Laboratory of Microbial Biochemistry, Kyoto University.
Takada
H
H
Yoshimura
T
T
Furuyoshi
S
S
Esaki
N
N
Ohshima
T
T
Soda
K
K
D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001483:Base Sequence; D002942:Circular Dichroism; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007700:Kinetics; D050803:Leucine Dehydrogenase; D008848:Micromonosporaceae / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D010649:Phenylalanine; D000594:Amino Acid Oxidoreductases; D050803:Leucine Dehydrogenase; C022342:phenylalanine oxidase