Photomodification of a serine at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase by vanadate.
Irradiation of ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach in the presence of vanadate at 4 degrees C resulted in rapid loss of carboxylase activity. The inactivation was light and vanadate dependent. When the enzyme was irradiated in the presence of the substrate ribulose 1,5-bisphosphate or an analogue such as fructose 1,6-bisphosphate, the inactivation was greatly reduced. Sodium bicarbonate and phosphate also protected against inactivation. No additional protection was observed in the presence of Mg2+ nor did Mg2+ alone protect. Carboxylase activity could be partially restored by treatment with NaBH4, and the photomodified protein could be tritiated with NaB3H4. Amino acid analysis showed that the tritium had been incorporated into serine. The data suggest that an active-site serine is photooxidized by vanadate to an aldehyde which results in activity loss. Irradiation in the presence of vanadate also resulted in cleavage in the large subunit of the enzyme which was subsequent to inactivation.
Mogel
S N
SN
Biochemistry/Biophysics Program, Washington State University, Pullman 99164-4660.
McFadden
B A
BA
D001894:Borohydrides / Q000494:pharmacology; D007700:Kinetics; D010600:Pharmacology; D010777:Photochemistry; D012273:Ribulose-Bisphosphate Carboxylase / Q000037:antagonists & inhibitors* / Q000528:radiation effects; D012694:Serine; D014466:Ultraviolet Rays
D001894:Borohydrides; D012694:Serine; D012273:Ribulose-Bisphosphate Carboxylase