Identification of active site lysyl residues of phenylalanine dehydrogenase by chemical modification with methyl acetyl phosphate combined with site-directed mutagenesis.
A monoanionic acetylation reagent, methyl acetyl phosphate, was used to acetylate lysyl residues of the recombinant thermostable phenylalanine dehydrogenase from Thermoactinomyces intermedius. The enzyme was irreversibly inactivated with the reagent in a time- and dose-dependent manner. Simultaneous addition of substrate and coenzyme markedly protected the enzyme from inactivation. Acetylated lysyl residues presumably occurring at the active site were determined by differential modification; the enzyme was first modified with a cold reagent in the presence of both substrate and coenzyme and, after removal of the added substances by gel filtration, was then labeled with a radioactive reagent. At least 7 lysyl residues per enzyme subunit were radiolabeled by this method. To further specify the lysyl residue(s) whose modification results in inactivation of the enzyme, 5 lysyl residues highly conserved in various amino acid dehydrogenase sequences were replaced with Ala by site-directed mutagenesis. Although all of the single mutant enzymes were inactivated with the reagent as effectively as the wild-type enzyme, a double mutant enzyme in which both Lys-69 and Lys-81 were replaced with Ala was found to be inactivated very slowly. These results suggest that the reagent can acetylate both of these lysyl residues and inactivate the enzyme. Kinetic analyses of the single Lys-69 and Lys-81 mutant enzymes revealed that they are involved in substrate binding and catalysis, respectively, like the corresponding residues in the homologous leucine dehydrogenase.
Kataoka
K
K
Laboratory of Microbial Biochemistry, Kyoto University.
Tanizawa
K
K
Fukui
T
T
Ueno
H
H
Yoshimura
T
T
Esaki
N
N
Soda
K
K
D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004789:Enzyme Activation; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D012680:Sensitivity and Specificity
C014660:methylacetylphosphonate; D000594:Amino Acid Oxidoreductases; C022342:phenylalanine oxidase; D008239:Lysine; D010746:Phosphonoacetic Acid