Active site modification of 4-aminobutyrate aminotransferase with ATP analogs

The dialdehyde of oxidized 1,N6-etheno-ATP and adenosine triphosphopyridoxal were used as probes of the catalytic site of 4-aminobutyrate aminotransferase. Both compounds react with lysine residues critically connected with aminotransferase activity. The binding of 1 mol of oxidized 1,N6-etheno-ATP per mol of enzyme or the binding of 1 mol of adenosine triphosphopyridoxal abrogates catalytic activity. The presence of substrate alpha-ketoglutarate (4 mM) prevents inactivation of the aminotransferase by either one of the ATP analogs. Reduction of the enzyme modified with oxidized 1,N6-etheno-ATP yields a chromophore which displays a maximum of emission at 415 nm and a fluorescent lifetime of 21.6 ns. The degree of exposure of the ethenoadenine ring to collisional encounters with the strong quencher KI was determined at pH 7.0. The ethenoadenine ring of the bound ligand is partially shielded from collisional encounters with the quencher. Steady-state emission anisotropy measurements of the bound ligand reveal that oxidized 1,N6-etheno-ATP is not rigidly attached to the protein matrix. It is postulated that the catalytic domain of 4-aminobutyrate aminotransferase is accessible to bulky reagents of greater length than the substrates 4-aminobutyrate and alpha-ketoglutarate.