Role of leucine 201 of thermostable D-amino acid aminotransferase from a thermophile, Bacillus sp. YM-1.
We studied the catalytic role of leucine 201 residue of the thermostable D-amino acid aminotransferase: the residue was shown crystallographically to be in the vicinity of the active site to interact with the bound pyridoxal phosphate. We replaced the leucine 201 by alanyl or tryptophanyl residues by means of site-directed mutagenesis. The L201A and L201W mutant enzymes showed anomalous kinetic behavior in the overall reaction. The reaction rates of the L201A and L201W mutant enzymes gradually decreased with an increase in the reaction time to become practically zero at a high concentration of substrates. The mutant enzymes were also inactivated in the half reaction with D-alanine, although more slowly than in the overall reaction. The absorption spectra of the mutant enzymes in the presence of D-alanine and alpha-ketoglutarate suggest that the enzyme molecules were mostly in the pyridoxamine form under the conditions employed. These phenomena were explained by assuming two (or more) enzyme species showing kinetically different catalysis for pyridoxamine form of the mutant enzymes, and the rate of conversion from one of these pyridoxamine forms to the pyridoxal form should be very low. The leucine 201 residue probably regulates the function of cofactor during the reaction of D-amino acid aminotransferase.
Kishimoto
K
K
Laboratory of Microbial Biochemistry, Kyoto University.
Yoshimura
T
T
Esaki
N
N
Sugio
S
S
Manning
J M
JM
Soda
K
K
D000409:Alanine / Q000378:metabolism; D000410:Alanine Transaminase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D051263:D-Alanine Transaminase; D004789:Enzyme Activation; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D007930:Leucine / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011733:Pyridoxamine / Q000031:analogs & derivatives / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism
D001426:Bacterial Proteins; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D014364:Tryptophan; D000410:Alanine Transaminase; D051263:D-Alanine Transaminase; D007930:Leucine; D000409:Alanine; C010627:pyridoxamine phosphate