Modification of carboxyl groups at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase.
Ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach was inactivated by a carboxyl-directed reagent, Woodward's reagent K ( WRK ). The inactivation followed pseudo-first-order kinetics. The reaction order with respect to inactivation by WRK was 1.1, suggesting that inactivation was the consequence of modifying a single residue per active site. The substrate ribulose 1,5-bisphosphate (RBP), two competitive inhibitors, fructose 1,6-bisphosphate (FBP) and sedoheptulose 1,7-bisphosphate (SBP), and a number of sugars-phosphate protected against inactivation by WRK . SBP was a strong protector, displaying a dissociation constant (Kd) of 3 microM with native RBP carboxylase. Pretreatment of RBP carboxylase with diethyl pyrocarbonate prevented WRK incorporation into the enzyme. The enol ester derivative produced by reaction of WRK with RBP carboxylase has a maximal absorbance at 346 nm, and the extinction coefficient was found to be 12300 +/- 700 M-1 cm-1. Spectrophotometric titration of the number of carboxyl groups modified by WRK in RBP carboxylase/oxygenase in the presence and in the absence of SBP suggests that inactivation was associated with the modification of one carboxyl group per active site.
D001665:Binding Sites; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D007202:Indicators and Reagents; D007555:Isoxazoles; D007700:Kinetics; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010944:Plants / Q000201:enzymology; D012273:Ribulose-Bisphosphate Carboxylase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013053:Spectrophotometry; D013379:Substrate Specificity
D007202:Indicators and Reagents; D007555:Isoxazoles; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D010105:Oxygenases; D012273:Ribulose-Bisphosphate Carboxylase