Mechanism of Binding of Pyridoxamine 5-Phosphate to the Apoenzyme Aspartate Aminotransferase

Abstract

The combination of pyridoxamine 5-phosphate with the apoprotein of the enzyme aspartate aminotransferase has been followed by measuring the quenching of protein fluorescence associated with complex formation. The reaction takes place in two steps; an initial rapid decrease in protein fluorescence is followed by a slow quenching process which parallels the recovery of catalytic activity. Similar delay in fluorescence decrease has been observed when pyridoxamine 5-phosphate fluorescence was immediately recorded after addition of apoprotein. These studies suggest that the slow decrease in fluorescence is associated with a change in protein conformation which is required for the formation of active species in solution. The interaction between pyridoxine 5-phosphate and the apoprotein was also examined by fluorescence spectroscopy and the results were compared with the inhibitory action of pyridoxamine 5-phosphate on the reconstitution of the holoenzyme.