Effect of substitution of a lysyl residue that binds pyridoxal phosphate in thermostable D-amino acid aminotransferase by arginine and alanine.
Lys-145 of the thermostable D-amino acid aminotransferase, which binds pyridoxal phosphate, was replaced by Ala or Arg by site-directed mutagenesis. Both mutant enzymes were purified to homogeneity; their absorption spectra indicated that both mutant enzymes contained pyridoxal phosphate bound non-covalently. Even though the standard assay method did not indicate any activity with either mutant, addition of an amino donor, D-alanine, to the Arg-145 mutant enzyme led to a slow decrease in absorption at 392 nm with a concomitant increase in absorption at 333 nm. This result suggests that the enzyme was converted into the pyridoxamine phosphate form. The amount of pyruvate formed was almost equivalent to that of the reactive pyridoxal phosphate in the mutant enzyme. Thus, the Arg-145 mutant enzyme is able to catalyze slowly the half-reaction of transamination. Exogenous amines, such as methylamine, had no effect on the half-reaction with the Arg-145 mutant enzyme. In contrast, the Ala-145 mutant enzyme neither underwent the spectral change by addition of D-alanine nor catalyzed pyruvate formation, in the absence of added amine. However, the Ala-145 mutant enzyme catalyzed the half-reaction significantly in the presence of added amine. These findings suggest that a basic amino acid residue, such as lysine or arginine, is required at position 145 for catalysis of the half-reaction. The role of the exogenous amines differs with various active-site mutant enzymes.
Nishimura
K
K
Institute for Chemical Research, Kyoto University, Japan.
Tanizawa
K
K
Yoshimura
T
T
Esaki
N
N
Futaki
S
S
Manning
J M
JM
Soda
K
K
D000409:Alanine; D000410:Alanine Transaminase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001120:Arginine; D001483:Base Sequence; D003001:Cloning, Molecular; D051263:D-Alanine Transaminase; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010957:Plasmids; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013053:Spectrophotometry; D013816:Thermodynamics
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D001120:Arginine; D000410:Alanine Transaminase; D051263:D-Alanine Transaminase; D008239:Lysine; D000409:Alanine