Activation of Ribulosebisphosphate Carboxylase/Oxygenase at Physiological CO₂ and Ribulosebisphosphate Concentrations by Rubisco Activase

Abstract

The enzyme-catalyzed activation of ribulosebisphosphate carboxylase/ oxygenase (rubisco) was investigated in an illuminated reconstituted system containing thylakoid membranes, rubisco, ribulosebisphosphate (RuBP), MgCl2, carbonic anhydrase, catalase, the artificial electron acceptor pyocyanine, and partially purified rubisco activase. Optimal conditions for light-induced rubisco activation were found to include 100 micrograms per milliliter rubisco, 300 micrograms per milliliter rubisco activase, 3 millimolar RuBP, and 6 millimolar free Mg2' at pH 8.2. The half-time for rubisco activation was 2 minutes, and was 4 minutes for rubisco deactivation. The rate of rubisco deactivation was identical in the presence and absence of activase. The K,,t(CO2) of rubisco activation in the reconstituted system was 4 micromolar C02, compared to a K,A,(C02) of 25 to 30 micromolar CO2 for the previously reported spontaneous CO2/Mg2' activation mechanism. The activation process characterized here explains the high degree of rubisco activation at the physiological concentrations of 10 micromolar CO2 and 2 to 4 millimolar RuBP found in intact leaves, conditions which lead to almost complete deactivation of rubisco in vitro.The photosynthetic assimilation ofCO2 is initiated by rubisco.2 This enzyme exhibits complex regulatory properties, including a requirement that it be converted to an activated state in order to acquire catalytic competency (8,9,14). The activation of rubisco in vitro occurs by the spontaneous addition of activating CO2 (ACO2) to the e-amino group of a lysine residue near the active site, followed by the addition of Mg2' (Eq. 1, where E = rubisco) (9, 10). (about 10 AM). Also, direct measurements have shown that rubisco activation in illuminated leaves is not very responsive to the intercellular CO2 concentrations (12, 17), although it does respond quantitatively over a wide range of light intensities (13,17). Thus the spontaneous mechanism of rubisco activation (9, 10) as shown in Eq. 1 is insufficient to explain the activation of the enzyme in vivo.We recently demonstrated (16) that the absence of rubisco activation in a mutant of the C3 plant Arabidopsis thaliana (20) was correlated with the absence of two chloroplast polypeptides, and that a soluble chloroplast extract stimulated rubisco activation in an illuminated reconstituted system containing chloroplast thylakoid membranes and rubisco. These observations led us to conclude that rubisco activation in vivo did not occur spontaneously but required the presence of an enzyme, rubisco activase. We have now partially purified the enzyme which catalyzes rubisco activation and demonstrate that it reduces the CO2 requirement for activation to a concentration consistent with that found in leaves.MATERIALS AND METHODS Chemicals and Equipment. RuBP was synthesized from ribose 5-P (Sigma3) as described previously (7) and stored in liquid N2. Some commercial preparations have been examined and found to be unsatisfactory for reconstitution studies. Pyocyanine...

Stimulation of thylakoid energization and ribulose‐bisphosphate carboxylase/oxygenase activation in Arabidopsis leaves by methyl viologen

“…By increasing the rate of CO2 fixation [15], higher concentations of CO2 dissipate the excess energy thus compensating for the limitation imposed by the inability of the system to activate rubisco. These findings suggest a central role for rubisco activase, not only in activating rubisco at physiological substrate levels [9], but also in coordinating the rates of CO2 fixation via changes in the activation state of rubisco with the rate of electrontransport activity.…”

“…Pyocyanine and MV, PS I electron-transport acceptors, were required for activation in the reconstituted system [8,9] and the latter stimulated rubisco light activation in vivo [3]. Rubisco activation in the well-buffered reconstituted assay (unpublished) and in vivo [4,10] is uncouplersensitive, suggestive of a requirement for a pH gradient across the thylakoid membrane.…”

“…We have recently shown that an association between thylakoids and rubisco activation can be reconstituted in vitro by the addition of rubisco activase, a soluble chloroplast protein [8], to purified rubisco and washed thylakoids in the presence of RuBP, ATP and light [8,9]. Pyocyanine and MV, PS I electron-transport acceptors, were required for activation in the reconstituted system [8,9] and the latter stimulated rubisco light activation in vivo [3].…”

Involvement of Stromal ATP in the Light Activation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Intact Isolated Chloroplasts

“…The activation state of rubisco increases with increasing light intensity, saturating at similar values to photosynthesis, whereas alkalization of the stroma and light-induced increases in stromal Mg2' are saturated at much lower light intensities (12,19). The K<, (CO2) for spontaneous activation of purified rubisco is 25 to 30 Mm (4, 7), which is three times higher than the CO2 concentration thought to exist in the chloroplast in vivo (13). Activation of purified rubisco is strongly inhibited by physiological concentrations of RuBP (4, 10).…”

The localisation of 2‐carboxy‐d‐arabinitol 1‐phosphate and inhibition of Rubisco in leaves of Phaseolus vulgaris L

“…The structural similarity of CA1P to the carboxylation transition state intermediate, 2-carboxy 3-ketoarabinitol 1,5-bisphosphate, strongly suggests a role in Rubisco regulation. The subsequent demonstration that both CA1P and a speci¢c phosphatase that degrades it were located in the chloroplast [5,6], and that CA1P could be released from Rubisco by Rubisco activase, gave further support to this hypothesis. In contrast, more recent claims by Anwaruzzaman et al [7] that CA1P and Rubisco are not in the same part of the leaf and that CA1P only becomes bound during extraction is inconsistent with a role for CA1P in Rubisco regulation and needs further examination.…”