Studies of the active-site lysyl residue of thermostable aspartate aminotransferase: combination of site-directed mutagenesis and chemical modification.
The gene technological substitution of the cysteinyl residue for the pyridoxal 5'-phosphate-binding lysyl residue (K239) of thermostable aspartate aminotransferase of Bacillus sp. YM-2 led to loss of the activity of the enzyme, which inherently contains no cysteinyl residues. The cysteinyl residue of the mutant enzyme was modified to lysine sulfur analog residues, S-(beta-aminoethyl)cysteine (SAEC), S-(beta-aminopropyl)cysteine (SAPC), and S-(beta-aminoethylthio)cysteine (SATC) with 2-bromoethylamine, 3-bromopropylamine, and 2-mercaptoethylamine, respectively. The modified mutant enzymes showed absorbance at 379 (K239SAEC), 400 (K239SAPC), and 365 nm (K239SATC), whereas the spectrum of the wild-type enzyme exhibited an absorption maximum at 360 nm derived from the internal Schiff base at pH 8.0. The absorption of the modified mutant enzymes at these wavelengths disappeared on reduction with NaCNBH3. This suggests that omega-amino groups of the introduced lysine sulfur analog residue form an internal Schiff base with pyridoxal 5'-phosphate. The modified mutant enzymes showed kcat values of 19.6-0.065% of that of the wild-type enzyme in the overall reaction, and were 10(6)-10(8) times more active than the K239C mutant enzyme. These results suggest that omega-amino groups of the introduced residues of the modified mutant enzyme serve as a catalytic base, and catalysis of the enzyme was affected by the length of the functional side-chain.
Kim
D W
DW
Laboratory of Microbial Biochemistry, Kyoto University.
Yoshimura
T
T
Esaki
N
N
Satoh
E
E
Soda
K
K
D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D004795:Enzyme Stability; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011732:Pyridoxal Phosphate / Q000032:analysis; D013329:Structure-Activity Relationship; D013696:Temperature
C000204:S-2-aminoethyl cysteine; D011732:Pyridoxal Phosphate; D001219:Aspartate Aminotransferases; D008239:Lysine; D003545:Cysteine