The lability of an intermediate of the ribulose bisphosphate carboxylase reaction.
Interruption of the catalytic cycle of ribulose-bisphosphate carboxylase by acid denaturation liberated an intermediate with a labile phosphate ester. Addition of fresh, buffered carboxylase enzyme to the acidified carboxylase reaction after 5 s inhibited phosphate release from the intermediate. Therefore, the species with a labile phosphate ester was stable for 5 s in acid and was apparently a substrate for the enzymatic reaction, since the labile intermediate was converted to a stable form by the protein. After acid denaturation, the carboxylated intermediate could be stabilized by reduction after 5 s in acid, but after 1 h no carboxylated intermediate remained. The stoichiometries of phosphate released to enzyme active sites and the carboxylated intermediate trapped to enzyme active sites were approximately 0.04. It was concluded that the labile phosphate species is probably the carboxylated intermediate rather than the enediol(ate) intermediate. The carboxylase and oxygenase reactions were probed for intermediates by the ability of the enzymatic reaction to reduce hexacyanoferrate(III), dichlorophenolindophenol, or nitroblue tetrazolium. Reduction of these reagents and hexacyanoferrate(III)-dependent paracatalytic inactivation were not observed. The copper chelate of lysine, a superoxide dismutase active species, did not selectively inhibit ribulose-bisphosphate oxygenase.
D001665:Binding Sites; D001894:Borohydrides / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D005634:Fructose-Bisphosphate Aldolase / Q000037:antagonists & inhibitors; D007700:Kinetics; D010761:Phosphorus Radioisotopes; D010944:Plants / Q000201:enzymology*; D011485:Protein Binding; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000378:metabolism
D001894:Borohydrides; D010761:Phosphorus Radioisotopes; D003300:Copper; C025364:sodium borohydride; D013482:Superoxide Dismutase; D012273:Ribulose-Bisphosphate Carboxylase; D005634:Fructose-Bisphosphate Aldolase