Gabaculine and m-carboxyphenyl-pyridoxamine 5-phosphate as probes of the catalytic binding sites of 4-aminobutyrate aminotransferase.
A homogeneous 4-aminobutyrate aminotransferase isolated from pig brain exhibits a k(cat) of 9.6 s-1 and contains one mole of pyridoxal 5-phosphate/mole of dimer. THe reaction of the enzyme with gabaculine (5-amino-1,3-cyclohexadiene carboxylic acid) was studied by observing changes in the absorption spectrum of the bound cofactor and by monitoring loss of catalytic activity. The enzyme is completely inactivated by gabaculine, but the dialyzed inactive sample containing 0.5 mol of gabaculine/mol dimer is fully reconstituted by addition of pyridoxal 5-phosphate. Stopped-flow kinetic studied reveal that gabaculine reacts with the cofactor bound to the aminotransferase with a second-order rate constant of 2.5 X 10(3) M-1 s-1. Fluorometric titrations of the apoprotein with m-carboxyphenyl-pyridoxamine 5-phosphate show the binding of two moles of inhibitor/mole of enzyme. The binding process is reversible and the affinity of the apoprotein for the inhibitor is at least 10-fold higher than the affinity for the cofactor. It is postulated that the dimeric enzyme contains two potential active sites per dinner, but the binding site characterized by a weaker affinity constant for pyridoxal 5-phosphate becomes functional only after specific chemical modification of the molecule of cofactor tightly bound to the protein.
D000612:4-Aminobutyrate Transaminase / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D003509:Cyclohexanecarboxylic Acids / Q000378:metabolism*; D003514:Cyclohexylamines / Q000378:metabolism; D007700:Kinetics; D011733:Pyridoxamine / Q000031:analogs & derivatives* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013552:Swine; D000637:Transaminases / Q000378:metabolism*
D003509:Cyclohexanecarboxylic Acids; D003514:Cyclohexylamines; C012987:gabaculine; C032422:3-carboxyphenylpyridoxamine 5-phosphate; D011733:Pyridoxamine; D000637:Transaminases; D000612:4-Aminobutyrate Transaminase