Temperature Dependence of the Enzymic Carboxylation and Oxygenation of Ribulose 1,5-Bisphosphate in Relation to Effects of Temperature

Carboxylase and oxygenase activities of ribulose bisphosphate carboxylase purified from wheat were measured over the temperature range 5 to 35°C either at constant 02 and CO2 concentrations or where the 02 and CO2 simulated the concentrations in water equilibrated at each temperature with the same gaseous phase. At constant CO2 (14 micromolar) and 02 (0.34 millimolar), the oxygenase to carboxylase ratio remained constant at 0.21 between 5 and 25°C but increased to 0.26 at 35°C. At 02 and CO2 concentrations near those expected in water equilibrated with air (21% Iv/ vl 02) containing 300 pI/I CO2 at the various temperatures, the ratio of oxygenase to carboxylase activity increased 2.2-fold between 15 and 35°C. At CO2 and 02 concentrations expected in water in equilibrium with subatmospheric concentrations of CO2 in air (21% Iv/vI 02, 210 id/i C02), the oxygenase to carboxylase ratio increased from 0.25 at 10°C to 0.56 at 35°C. Between 20 and 30°C, the apparent Qlo value for the oxygenase reaction was 1.78 and that for the carboxylase was 1.26. Hence, the different responses of photosynthesis and photorespiration to temperature are due more to changes in the relative solubilities of CO2 and 02 (the solubility ratio) than to changes in kinetic parameters of the reactions catalyzed by ribulose bisphosphate carboxylase. In C3 plants, 02 inhibition of photosynthesis, the CO2 compensation point, glycolate synthesis, and photorespiration are all increased with increasing temperature, more -rapidly than net photosynthesis (12, 13, 15, 26) and a corresponding decrease in quantum efficiency is found (8). Attempts have been made to explain these observations in terms of the properties of RuBP1 carboxylase/oxygenase (EC 4.1.1.3.9). Thus, if it is the oxygenase activity of RuBP carboxylase that results in synthesis of most of the substrate for photorespiration, namely phosphoglycolate (19), an increase in the oxygenase to carboxylase velocity ratio (v0/v,) in vivo with increased temperature could explain the observed effects on photorespiration and photosynthesis. The velocity ratio v0/v, was reported to increase with temperature (2, 17) and the change was considered sufficient to account for increased photorespiration relative to photosynthesis. Studies of the enzyme isolated from C3 plants showed that the kinetic parameters of the oxygenase and carboxylase activities change differently as the temperature increases and this may explain changes in v0/v, and hence increased photorespiration (3, 17). Another proposal is that the increased solubility ratio 02/CO2 with increasing temperature may be a major cause of the increased 'Abbreviation: RuBP, ribulose 1,5-bisphosphate. photorespiration (16). Tenhunen et al. (24) have expressed doubts on this conclusion and have urged consideration of an empirical model. Badger and Collatz (3) proposed the use ofpartial pressures of CO2 and 02 in the gas phase as more appropriate measures than concentration in solution of the activities of CO2 and 02 in solution. However, treatments of enzyme kinetics conventionally use concentrations in solution and it is not helpful to introduce the alternative of partial pressures unless in a thorough treatment in terms of activities involving assessment of fugacities for the gaseous reactants. Calculations based on the data of Badger and Collatz (3) in terms of concentrations of CO2 and 02 in solution, compared to use of partial pressures ofCO2 and 02 in equilibrium, confirm the significance of solubility ratio as the main cause of increase of photorespiration to photosynthesis with temperature. Reported kinetic parameters ofRuBP carboxylase/oxygenase and the intercellular partial pressures of CO2 and 02 have been incorporated into mathematical models of photosynthesis and photorespiration, which also take into account limitations by the light reactions (9). It is important to determine whether the reaction kinetics based on concentrations of substrates in solution or gas solubility ratio is the most significant feature determining the effect of temperature on photosynthesis and photorespiration in order to make the models more accurate. This paper describes the direct measurement in the same reaction mixtures of carboxylase and oxygenase activities under various defined conditions oftemperature CO2 and 02 concentrations, using fully activated enzyme. This approach was used since measurement of the kinetic constants is complicated by the fact that the oxygenase reaction is not saturated at 100%02 at atmosphere pressure (1). The objective was to determine how v0/v, changes with temperature at constant concentrations of CO2 and 02, and hence where only changes in conventional parameters of enzyme kinetics are involved, as well as under conditions designed to simulate those expected in the chloroplast stroma where relative concentrations of gaseous substrates are also changed with temperature because of changes in solubility. MATERIALS AND METHODS RuBP Carboxylase from Wheat Leaves. The purified enzyme was available as a freeze-dried powder (5) and was activated by incubation for 1 h at 35°C in 100 mm Bicine-NaOH (pH 8.2) containing 10 mm NaHCO3 and 20 mm MgCl2. Subsequently, the enzyme solution was maintained at 25°C for the duration of the experiment. The specific activity of the preparations used was 0.5 to 0.9 ,umol/min-mg protein at 25°C and 5 mm bicarbonate. Preparation of Buffers. Bicine and MgCl2 were added to distilled H20 that had been boiled and then cooled under N2. N2 was bubbled through the solution for approximately 1 h before adjusting to the final pH with 'CO2-free' NaOH (BDH). The solution