Pyridoxal phosphate as a probe in the active site of ribulose bisphosphate carboxylase/oxygenase.
Ribulose bisphosphate (RuBP) carboxylase is rapidly and irreversibly inactivated by photooxidation sensitized by pyridoxal phosphate. Both pyridoxal and pyridoxamine phosphate were much less effective in sensitizing the photooxidation even when used at twice the concentration of pyridoxal phosphate. These results imply that pyridoxal phosphate binds at the active site not only through a Schiff base, but also through ionic interaction with the phosphate binding region. Spectral analysis of the photooxidized enzyme showed a new absorption maximum at 325 nm due to reduction of the Schiff base between pyridoxal phosphate and a lysyl residue with concomitant oxidation of a histidine residue. The stoichiometry of photooxidative [3H]pyridoxal phosphate incorporation was 0.87 mol/mol of a 70,000-dalton large subunit-small subunit combination. Studies with 3H-labeled diethyl pyrocarbonate showed that both photooxidation and carbethoxylation occur at the same histidine residue. However, photooxidation by pyridoxal phosphate is very specific for an active site histidine residue due to the high specificity of this affinity label. Several competitive inhibitors with respect to ribulose bisphosphate offered appreciable protection against pyridoxal phosphate-induced photooxidation of the enzyme. The photooxidized enzyme showed an increase in the net negative charge on the protein which was evident from the higher mobility of the photooxidized enzyme toward the anode in polyacrylamide gel electrophoresis.
D001665:Binding Sites; D002262:Carboxy-Lyases; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine; D008239:Lysine; D010084:Oxidation-Reduction; D010777:Photochemistry; D010944:Plants; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D012273:Ribulose-Bisphosphate Carboxylase; D013057:Spectrum Analysis
D006639:Histidine; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D002262:Carboxy-Lyases; D012273:Ribulose-Bisphosphate Carboxylase; D008239:Lysine