Evidence for a carbanion intermediate in catalysis by spinach ribulose-1,5-bisphosphate carboxylase/oxygenase

Spinach ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (EC 4.1.1.39) showed marked reactivity towards tetranitromethane in the presence of RuBP, when the enzyme was in the fully activated state. The inactivated enzyme did not catalyze substrate-dependent nitroform production. Nitroform production was proportional to the concentration of the enzyme and tetranitromethane at optimal substrate concentrations. The pH optimum for nitroform production was similar to that for carboxylation. The effects of bicarbonate, ribulose-1,5-bisphosphate, and temperature on partition of the intermediate between production of phosphoglycerate and nitroform indicate that both routes share a common intermediate. Data on stoichiometry of the reaction imply that the intermediate is not oxidized by tetranitromethane but instead is nitrated. The data are compatible with a carbanion intermediate in the reaction catalyzed by ribulose-1,5-bisphosphate carboxylase.