Activities | Families | Sequences | Fold types | References | Help
B6db references: 97201073

type Journal Article
authors Kim, Y. T.; Song, Y. H.; Churchich, J. E.
title Recombinant brain 4-aminobutyrate aminotransferases overexpression, purification, and identification of Lys-330 at the active site
journal Biochim Biophys Acta
ui 97201073
year (1997)
volume 1337
number 2
pages 248-56.
keywords 4-Aminobutyrate Transaminase/chemistry/*genetics/*isolation & purification
abstract 4-Aminobutyrate aminotransferase (4-aminobutyrate: 2-oxoglutarate aminotransferase EC is a key enzyme of the 4-aminobutyric acid shunt. It catalyzes the conversion of 4-aminobutyrate to succinic semialdehyde. In an effort to clarify the structure-function relationships of 4-aminobutyrate aminotransferase, we analyzed 4- aminobutyrate aminotransferase cDNA from pig brain. The inclusion bodies were formed when recombinant 4-aminobutyrate aminotransferase was overexpressed in Escherichia coli. The unfolded overproduced proteins, were purified by hydroxylapatite chromatography in the presence of urea and refolded by a sequential dialysis method. The renatured protein regained its catalytic activity. The lysyl residue at the 330 position of the amino-acid sequence serves as the anchoring site of the cofactor pyridoxal 5'-P. To verify the catalytic site of 4- aminobutyrate aminotransferase, lysine 330 was mutated to arginine by site-specific mutagenesis. Overexpression and purification of the mutated 4-aminobutyrate aminotransferase (K330R) were performed by the same method used the purification of wild-type 4-aminobutyrate aminotransferase. The purified and renatured K330R protein did not show the catalytic activity of wild type 4-aminobutyrate aminotransferase. Furthermore, the mutated protein did not show any absorption band over the spectral range of 320-460 nm characteristic of pyridoxal 5'-P covalently linked to the protein. From the results presented here, it is concluded that lysine 330 is essential for the catalytic function of the aminotransferase.
last changed 2002/11/04 17:41

B6db references