Inhibition of ribulose bisphosphate carboxylase by substrate ribulose 1,5-bisphosphate.
Substrate ribulose bisphosphate is a potent and a weak inhibitor of the rate of CO2/Mg2+ activation in the carboxylase purified from spinach leaves and Rhodospirillum rubrum, respectively. At 2 degrees C, the concentration of ribulose bisphosphate required for 50% inhibition of the initial rate of CO2/Mg2+ activation was less than 0.4 microM for the spinach enzyme, but between 67 and 270 microM for the R. rubrum carboxylase. Activator 14CO2 trapping experiments demonstrated that ribulose bisphosphate inhibits activation by excluding activator CO2 from the spinach enzyme. The reason for the different sensitivities to inhibition by substrate was evident from equilibrium binding studies with the inactive enzyme forms which indicated that the KD (ribulose bisphosphate) was 0.021 microM for spinach enzyme and 5.9 microM for the R. rubrum protein. Inhibition of activation, however, was not explained by the equilibrium binding results alone. Ribulose bisphosphate was observed to dissociate very slowly from the inactive spinach enzyme (at 2 degrees C, kOFF = 4.9 X 10(-5) s-1). The release of substrate from the inactive R. rubrum carboxylase was much more rapid, with a minimum value for kOFF estimated at 5 X 10(-3) s-1 at 2 degrees C. We conclude that strong inhibition of CO2/Mg2+ activation in the spinach enzyme is mediated by the tight binding and slow release of ribulose bisphosphate, which prevent activator CO2 and Mg2+ from binding to the protein. Weak inhibition of activation in the R. rubrum enzyme results from a larger KD value and a more rapid exchange of ribulose bisphosphate, which allow activator CO2 and Mg2+ to bind to the free enzyme between successive substrate-binding events.
D002245:Carbon Dioxide / Q000494:pharmacology; D004789:Enzyme Activation; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010428:Pentosephosphates / Q000494:pharmacology*; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000037:antagonists & inhibitors*; D012274:Ribulosephosphates / Q000494:pharmacology*
D010428:Pentosephosphates; D012274:Ribulosephosphates; D002245:Carbon Dioxide; C001933:ribulose-1,5 diphosphate; D012273:Ribulose-Bisphosphate Carboxylase; D008274:Magnesium