RECOMBINANT BRAIN 4-AMINOBUTYRATE AMINOTRANSFERASES OVEREXPRESSION, PURIFICATION, AND IDENTIFICATION OF LYS-330 AT THE ACTIVE-SITE

4-Aminobutyrate aminotransferase (4-aminobutyrate: 2-oxoglutarate amin otransferase EC 2.6.1.19) is a key enzyme of the 4-aminobutyric acid s hunt. It catalyzes the conversion of 4-aminobutyrate to succinic semia ldehyde. In an effort to clarify the structure-function relationships of 4-aminobutyrate aminotransferase, we analyzed 4-aminobutyrate amino transferase cDNA from pig brain. The inclusion bodies were formed when recombinant 4-aminobutyrate aminotransferase was overexpressed in Esc herichia coli. The unfolded, overproduced proteins, were purified by h ydroxylapatite chromatography in the presence of urea and refolded by a sequential dialysis method. The renatured protein regained its catal ytic 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, lys ine 330 was mutated to arginine by site-specific mutagenesis. Overexpr ession and purification of the mutated 4-aminobutyrate aminotransferas e (K330R) were performed by the same method used the purification of w ild-type 4-aminobutyrate aminotransferase. :The purified and renatured K330R protein did not show the catalytic activity of wild-type 4-amin obutyrate aminotransferase. Furthermore, the mutated protein did not s how any absorption band over the spectral range of 320-460 nm characte ristic of pyridoxal 5'-P covalently linked to the protein. From the re sults presented here, it is concluded that lysine 330 is essential for the catalytic function of the aminotransferase.