Activity and spectroscopic properties of bacterial D-amino acid transaminase after multiple site-directed mutagenesis of a single tryptophan residue.
One of the three tryptophan residues per subunit of thermostable D-amino acid transaminase, Trp-139, is close to the active-site Lys-145 in the sequence of the protein. This tryptophan has been changed to several other types of residues by site-directed mutagenesis. The only mutant protein that was sufficiently active and stable for study had Phe substituted for Trp (W139F). The spectroscopic properties of this mutant enzyme differed from those of the wild-type transaminase. For example, denatured W139F showed the expected decrease in fluorescence emission intensity at 350 nm due to the deletion of one Trp residue, but the fluorescence emission of the wild-type and W139F enzymes in the native state did not differ in intensity. This result suggests that the fluorescence of Trp-139 in the native, wild-type enzyme is not manifested perhaps due to its proximity to the coenzyme, pyridoxal phosphate. Results of energy-transfer studies at several wavelengths could also be interpreted as due to the proximity of Trp-139 and the coenzyme. Circular dichroism studies indicated that the negative Cotton effect at 420 nm due to the coenzyme was still present in W139F. However, the 280-nm optically active band present in the wild-type enzyme was greatly diminished in W139F. The mutant protein with Asp at position 139 (W139D) could not be isolated presumably because it was degraded. The other mutant enzymes, W139P, W139A, and W139H, were isolated with partial activities (15-35%) that were slowly lost upon storage at 4 degrees C. Overall, these results indicate the importance of Trp-139 in the thermostable D-amino acid transaminase.
Martínez del Pozo
A
A
Rockefeller University, New York, New York 10021.
Merola
M
M
Ueno
H
H
Manning
J M
JM
Tanizawa
K
K
Nishimura
K
K
Asano
S
S
Tanaka
H
H
Soda
K
K
Ringe
D
D
D001407:Bacillus / Q000201:enzymology*; D002942:Circular Dichroism; D051263:D-Alanine Transaminase; D007700:Kinetics; D046911:Macromolecular Substances; D009154:Mutation; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D000637:Transaminases / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan
D046911:Macromolecular Substances; D014364:Tryptophan; D000637:Transaminases; D051263:D-Alanine Transaminase