Determination of Apparent Km Values for Ribulose 1,5- Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Using the Spectrophotometric Assay of Rubisco Activity1

The spectrophotometric assay for ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) was used to determine the rate of increase in Rubisco activity over time in the presence or absence of Rubisco activase. Polynomial approximations to the raw data were used to smooth out minor fluctuations in the spectrophotometer readings, and Rubisco activase activity was expressed as nanomoles of activated Rubisco per minute. This assay was used to examine the effects of CO2 and the inacfiveRubisco:ribulose 1,5-bisphosphate complex (ER) on the activasecatalyzed activation reaction. Double-reciprocal plots of activase activity and ER at several concentrations of CO2 were consistent with two-substrate Michaelis-Menton kinetics, and the apparent Km(CO2) and Km(ER) were determined to be 53 and 2.7 micromolar, respectively. These data do not prove that ER and CO2 are substrates for the reaction catalyzed by activase, but they may be important to our understanding of the activation process in vivo. The implications of these data and their relation to previously published data on the effects of ER and CO2 on activase are discussed. In many plants, extractable Rubisco2 activity is low for leaves in low light, but increases when the leaf is transferred to a higher light intensity. This increase in activity occurs over a period of minutes and can limit the rate of photosynthesis during the nonsteady-state following an increase in light intensity (16, 21). For plants without the tight-binding inhibitor carboxyarabinatol 1-P, the increase in Rubisco activity associated with higher light intensities is the result of covalent modification of the catalytic site by CO2 and Mg2+. This reaction involves the formation of a carbamate at a specific lysine residue, which is then stabilized by Mg2", to form the catalytically competent enzyme (9). The thermodynamic equilibrium of the activation reaction, however, favors the noncarbamylated (inactivated) form of the enzyme under the conditions thought to exist in the stroma of an illuminated chloroplast. Tight binding of the substrate RuBP to the inactivated form of the enzyme further increases the proportion of the enzyme in the inactivated form (7), and the kff for this ' Supported by U.S. Department of Agriculture/Cooperative State Research Service grant 89-37130-4741. 2 Abbreviations: Rubisco, ribulose 1,5-bisphosphate carboxylase/ oxygenase; RuBP, ribulose 1,5-bisphosphate; ER, inactive-Rubisco:RuBP complex; ECM, activated Rubisco. binding restricts the rate at which active enzyme can be formed from the ER in vitro (10). Inactivated enzyme has been shown to bind to RuBP in intact leaves (1, 3), and it is therefore impossible to explain the relatively rapid activation rates and high steady-state activation states that are observed in leaves based on the activation process as studied in vitro. The discovery of the enzyme Rubisco activase (15), which apparently catalyzes some aspect of the activation reaction, has provided an explanation for the rapid activation rates observed in intact leaves. The discovery that the activasecatalyzed reaction also dephosphorylates ATP (18) has explained how a substantial portion of Rubisco can exist in the activated form in intact leaves despite the unfavorable equilibrium of the noncatalyzed reaction. A hypothetical reaction for Rubisco activase, along with the uncatalyzed activation reactions, is shown below, where E is Rubisco, R is RuBP, C is C02, and M is Mg2+: R R + + M + C + E ± ECM ATP + M + C + ER ± ECMR + ADP + Pi activase Since the increase in Rubisco activity following an increase in photon flux density has been shown to limit the approach of photosynthesis to steady-state following an increase in photon flux density (16, 21), the kinetics of the activasecatalyzed reaction may be important for the total carbon gain of plants growing in fluctuating-light conditions. In this study, we used the spectrophotometric assay of Rubisco activity (8) to monitor increases in Rubisco activity over time in the presence of Rubisco activase. The dependence of activase activity on CO2 and ER was examined and found to be consistent with two-substrate, Michaelis-Menton kinetics. MATERIALS AND METHODS