4-Aminobutyrate aminotransferase. The presence of nonequivalent binding sites

A homogeneous 4-aminobutyrate aminotransferase isolated from pig brain exhibits a kcat = 9.6 s-1 and contains only 1 mol of pyridoxal-5-P/mol of dimer. The equilibrium dissociation constant for pyridoxal-5-P tightly bound to the enzyme is 1 nM. Spectrophotometric titrations reveal that the enzyme binds a second molecule of pyridoxal-5-P with a KD = 3 microM. The reaction of enzyme containing 1 pyridoxal-5-P/dimer with the inhibitors NaBH4 and DL-gabaculine was studied by observing changes in the absorption spectrum of the bound coenzyme and by monitoring loss of catalytic activity. The native enzyme is inactivated by both inhibitors. Incubation of the resultant P-pyridoxyl aminotransferase and m-anthraniyl-PMP-aminotransferase with excess pyridoxal-5-P at 25 degrees C restores full catalytic activity. It is postulated that the dimeric enzyme contains two classes of catalytic binding sites, and that the binding site characterized by a weak affinity for pyridoxal-5-P (KD = 3 microM) becomes functional after specific chemical modification of the molecule of cofactor tightly bound to the protein (KD - 1 nM).