Pyridoxal 5′ –phosphate as photosensitizer in photo inactivation of multienzyme complexes

The reaction of pyridoxal 5′ phosphate(PLP) with fatty acid synthetase(FAS) from pigeon liver both in the presence and absence of light resulted in inactivation of overall FAS activity. Under both conditions similar kinetics of inactivation was observed for the first 8 min. NADPH showed a 60% of protection of the illuminated sample suggesting that modification of the essential residues is involved in the binding of the coenzyme. Although the PLP‐treated enzyme in the dark was reactivated by subsequent dilution with the buffer but the illuminated enzyme sample was not reactivated on dilution. Spectrophotometric examination of the pyridoxal‐phosphate treated fatty acid synthetase in the light and in dark suggested that the environment of PLP bound to the enzyme is altered by a nearby group resulting in appearance of a new fluorescence maxima at 445 nm when excited at 325 nm in the absence of reduction with sodium borohydride. This observation indicated that PLP and lysine of an active site of FAS formed a schiff base which has undergone an attack by a nearby nucleophilic group resulting in an irreversible and inactive enzyme‐PLP complex. It appears that imidazole group of a histidine residue may be the nucleophilic agent involved in the photo‐modification. These studies with pigeon liver FAS showed that lysine and histidine residues are present in enoyl –CoA reductase domain.