Fluorescence Studies on the Interaction between Pyridoxal Phosphate Enzymes

The association of the enzyme cystathionase with the apoprotein of the enzyme aspartate aminotransferase was investigated by fluorescence spectroscopy at protein concentrations lower than 1 mg per ml. The interaction between cystathionase and the 4-pyridoxic-5'-P-apoaminotransferase complex at pH 7.4 brings about dissociation of the inhibitor 4-pyridoxic-5'-P from the catalytic site of the aminotransferase. The rate of release of 4-pyridoxic-5'-P is easily monitored by measuring its increase in fluorescence at 425 nm as a function of incubation time. The rate of fluorescence enhancement at 425 nm, which is related to the dissociation of 4-pyridoxic-5'-P from the apoaminotransferase, is influenced by the molar ratio of cystathionase to aminotransferase in the reaction mixture. The resolved form of the enzyme cystathionase failed to dissociate the 4-pyridoxic-5'-P-apoaminotransferase complex, while free pyridoxal-5-P is much less efficient than pyridoxal-5-P bound to the cystathionase in promoting dissociation of the inhibitor 4-pyridoxic-5'-P. Polarization of fluorescence measurements on aspartate aminotransferase labeled with fluorescein isothiocyanate was used to detect the formation of a complex of this enzyme with cystathionase. The binding of labeled transaminase to cystathionase is accompanied by an increase in the polarization of fluorescence from 0.21 to 0.30 without any change in the fluorescence life-time of the fluorophore (τ = 4.5 ns) covalently bound to the aminotransferase. The binding process is reversible and the results of the polarization titrations indicate a strong affinity of the aminotransferase for cystathionase (Kd = 1 µm).