Interaction of sugar phosphates with the catalytic site of ribulose-1,5-bisphosphate carboxylase.
The activated and catalytically competent form of ribulose-1,5-bisphosphate carboxylase is a ternary complex of enzyme-activator CO2 x Mg. The effectors of NADPH and 6-phosphogluconate promoted activation by formation of a rapid equilibrium quaternary complex of enzyme-activation CO2 x Mg x effector; i.e., the effectors did not activate the enzyme per se but promoted the basic activation process by stabilizing the activated enzyme-activator CO2 x Mg complex. Kinetic and gel filtration studies showed that the effectors stabilized the binding of the activator CO2 and MG2+ (or Mn2+), thereby decreasing the rate of deactivation. Binding studies indicated the presence of one 6-phosphogluconate binding site per protomer. The binding of 6-phosphogluconate and NADPH to the enzyme-activator CO2 x Mg complex was (a) completely prevented when the catalytic site for ribulose bisphosphate was occupied by the transition-state analogue, 2-carboxyarabinitol 1,5-bisphosphate, and (b) competitively diminished in the presence of 3-phosphoglycerate, the product of the carboxylation reaction. NADPH, 6-phosphogluconate, and 3-phosphoglycerate acted as linear competitive inhibitors of carboxylation with respect to ribulose bisphosphate. These results demonstrate that the effectors elicit their response through interaction at the catalytic site for ribulose bisphosphate and that their effect is secondary to the basic CO2-Mg2+-dependent activation reaction. An enzyme molecule cannot be simultaneously catalytically competent (capable of binding and carboxylating ribulose bisphosphate) and activated by an effector, since the latter involves occupancy of the ribulose bisphosphate binding site.
D001665:Binding Sites; D002262:Carboxy-Lyases / Q000378:metabolism*; D004163:Diphosphoglyceric Acids / Q000494:pharmacology; D004789:Enzyme Activation; D005942:Gluconates / Q000494:pharmacology; D007700:Kinetics; D009249:NADP / Q000494:pharmacology; D010944:Plants / Q000201:enzymology; D011485:Protein Binding; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism*; D064030:S100 Calcium Binding Protein G / Q000494:pharmacology; D013403:Sugar Phosphates / Q000494:pharmacology
D004163:Diphosphoglyceric Acids; D005942:Gluconates; D064030:S100 Calcium Binding Protein G; D013403:Sugar Phosphates; D009249:NADP; D002262:Carboxy-Lyases; D012273:Ribulose-Bisphosphate Carboxylase; C008884:6-phosphogluconic acid