Photoinactivation of Aspartate Aminotransferase

The cofactor pyridoxal 5'-phosphate bound through an aldimine linkage to lysine residues of the enzyme aspartate aminotransferase is very stable to irradiation with light of 420 nm. The catalytic function of the enzyme exposed to light absorbed by the cofactor remains unaffected, indicating that pyridoxal 5'-phosphate is not an efficient active site photosensitizer for this aminotransferase. The irradiation of the apoprotein in the presence of lumichrome leads to irreversible loss of catalytic activity which follows first order kinetics. Photoinactivation of the apoprotein occurs at low concentrations of lumichrome (molar ratio of lumichrome to apoenzyme, 15:1), even though lumichrome is not bound to acceptor sites of the protein as demonstrated by spectrophotometric and fluorometric titrations. It is postulated that the mechanism underlying the photodynamic action of lumichrome must be operative over large distances. Transfer of excitation energy from the excited state (singlet or triplet) of the sensitizer lumichrome to corresponding excited states in the protein is extremely unlikely. An analysis of the photoinactivation results indicates that singlet excited oxygen, generated by energy transfer from the lowest triplet of lumichrome to oxygen, may act as the oxidizing agent of the histidine residues of the protein.