Linearity and Functioning Forms in the Carboxylase Reaction of Spinach Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase1

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The reaction of spinach RuBisCO activated with CO2 and Mg2+ proceeded in two phases, an initial burst for a few minutes and the subsequent linear phase, in the presence of saturating concentrations of CO2, ribulose 1,5-bisphosphate (RuBP), and Mg2+. The percentage of the activity in the linear phase to that in the initial burst was 55% with RuBisCO prepared with polyethylene glycol, and very close to the value with the enzyme released immediately from isolated chloro-plasts. RuBisCO prepared with ammonium sulfate had a much larger decrease of the activity in the linear phase. The Euglena enzyme had a linear course of reaction with time for up to 20 minutes. The Km for CO2 of spinach RuBisCO activated beforehand was 20 μM in the initial burst, and 28 μM in the linear phase. In the carboxylase reaction initiated with inactive enzyme, the activity was initially negligible, but in 5 minutes increased to the level observed in the linear phase of the activated enzyme. The Km for CO2 in the linear phase of the pre-inactivated enzyme was 70 μM. The concentration of RuBP was the immediate cause of the two-phasic course of the carboxylase reaction of spinach RuBisCO. The curvature of the time course was not observed below 35 μM RuBP. The enzyme required over 88 μM RuBP for the conventional two-phasic course. Further increase of the concentration of RuBP increased the extent of the curvature, but did not start the curvature sooner after the start of the reaction. Even if spinach RuBisCO was in the linear phase, dilution of RuBP or its consumption by the enzymatic reaction to less than 30 μM caused the enzyme to show the resumed biphasic reaction course after addition of a high concentration of RuBP.