Modification of carboxyl groups at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase

Abstract

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 ( K d ) of 3 μ m 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.