Evidence that Ribulose 1 , 5 - Bisphosphate ( RuBP ) Binds to Inactive Sites of RuBP Carboxylase in Vivo and an Estimate of the Rate Constant for Dissociation ' Zoe

The binding of ribulose 1,5-bisphosphate (RuBP) to inactive (noncarbamylated) sites of the enzyme RuBP carboxylase in vivo was investigated in Spinacla oleracea and Helianthus annuus. The concentrations of RuBP and inactive sites were determined in leaf ffssue as a function of time after a change to darkness. RuBP concentrations fell rapidly after the change to darkness and were approximately equal to the concentration of inactive sites after 60 s. Variations in the concentration of inactive sites, which were induced by differences in the light intensity before the light-dark transition, correlated with the concentration of RuBP between 60 and 120 s after the change to darkness. These data are discussed as evidence that RuBP binds to inactive sites of RuBP carboxylase In vivo. After the concentration of RuBP fell below that of inactive sites (at times longer than 60 s of darkness), the decline in RuBP was logarithmic with time. This would be expected if the dissociation of RuBP from inactive sites controlled the decline in RuBP concentration. These data were used to estimate the rate constant for dissociation of RuBP from inactive sites In vivo. Ribulose 1,5-bisphosphate carboxylase catalyzes the fixation of atmospheric CO2 in the photosynthetic carbon reduction cycle. It is now well established that this enzyme has eight active sites per holoenzyme and that each site is not catalytically competent unless complexed with CO2 and Mg2'. The addition of CO2 and Mg2' to a site, termed activation, involves the carbamylation of a lysine residue, and the percentage ofactivated sites (activation state) can be manipulated in vitro by varying the concentrations of CO2 and Mg2' (9). The mechanism of control for activation state in vivo is, however, still controversial; it is unlikely that changes in stromal CO2 and Mg2" concentrations are large enough to cause the variation in activation state observed. In many plants, the activity of RuBP2 carboxylase is regulated by the state of activation. However, a tight-binding inhibitor of RuBP carboxylase, carboxyarabinatol 1-phosphate, has been implicated in the control of RuBP carboxylase activity in some plants, independent of changes in activation state (16). 'Supported by National Science Foundation grant DMB-85 15578 and Utah State Agricultural Experiment Station Project 544. Published as Utah Agricultural Experiment Station Journal publication No. 3601. 'Abbreviations: RuBP, ribulose 1,5-bisphosphate; CABP, 2-carboxyarabinatol bisphosphate. 1253 In vitro studies with RuBP carboxylase have demonstrated that the substrate RuBP binds tightly to inactive (noncarbamylated) sites on the enzyme, excluding the activator CO2 molecule, and thus preventing activation ofthe site (8). RuBP carboxylase, therefore, activates very slowly in the presence of RuBP, even at high concentrations of CO2 and Mg2'. The tight binding of RuBP to inactive sites also stabilizes the inactive form of the enzyme and shifts the activation equilibrium toward deactivation (7). Thus, RuBP substantially inhibits the rate of activation and will deactivate previously activated enzyme in vitro. The binding of RuBP to inactive RuBP carboxylase poses questions concerning the control of activation state in vivo. In contrast to in vitro studies, activation proceeds relatively rapidly in vivo in response to an increase in light intensity (13), and is essentially complete in 5 min. Furthermore, the enzyme has been shown to achieve high activation states (approaching 100%) at high light intensities when RuBP concentrations are typically 4 mm or higher. Several hypotheses have been advanced to explain the activation and maintenance of high activation states ofRuBP carboxylase in vivo. Mott and Berry (11) found that the apparent Acd for the binding of RuBP to inactive sites in vitro increased at high pH values. Activation proceeded rapidly in the presence of high concentrations of RuBP, and high activation states were attainable at air-level CO2 concentration at pH values of 8.6 or higher. However, there is no evidence that steady state pH values of the stroma reach this value, even at saturating light intensity. Portis et al. (15) showed that activation in vitro occurred at high RuBP concentrations in the presence ofan enzyme extracted from chloroplasts, which they called RuBP carboxylase activase. Activity of RuBP carboxylase activase requires ATP (17), but its mechanism and role in regulating activation state in vivo have not been defined. Both ofthese hypotheses assume that RuBP binds to inactive sites of RuBP carboxylase in vivo, but the only data supporting this are those of Brooks and Portis (3), who reported that the amount of protein-bound RuBP varied in parallel with the percentage of inactive RuBP carboxylase sites. In this study, changes from light to dark were used to investigate the binding of RuBP to inactive sites of RuBP carboxylase in intact leaves. RuBP concentrations and RuBP carboxylase activation state were measured both in the illuminated steady state and as a function of time after a step change to darkness. The photosynthetic carbon reduction www.plantphysiol.org on July 27, 2017 Published by Downloaded from Copyright © 1989 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 89, 1989 cycle should not generate RuBP during the dark period, and active RuBP carboxylase should rapidly consume all remaining free RuBP. RuBP bound to inactive sites of RuBP carboxylase should not be immediately available to the activated sites, but this pool should disappear slowly as RuBP dissociates from the inactive sites and reacts at activated sites. The time-dependent disappearance of RuBP following the lightdark transition is discussed as evidence for binding of RuBP to inactive sites of RuBP carboxylase in vivo, and a rate constant for dissociation is estimated. MATERIALS AND METHODS Sunflower (Helianthus annuus) was grown in sterile potting soil that was watered with nutrient solution, and spinach (Spinacia oleracea) was grown either as above or hydroponically. Both species were grown in controlled environment growth chambers in which the light intensity at the top of each plant was maintained at approximately 350 ME(m2s)-'. The photoperiods for spinach and sunflower were 10 and 15 h, respectively. Gas Exchange and Quick-Kill For experiments that required low CO2 treatment, leaves were placed in a clamp-on leaf chamber, and gas exchange parameters were measured using a gas mixing and analysis system (10). When the appropriate conditions existed in the chamber, the leaf was quick-killed by firing a stainless steel cutting tube cooled with liquid nitrogen through the chamber with an air driven piston. The top and bottom ofthe chamber were constructed of paraffilm, and they were cut with the leaf tissue and clamped between the bottom of the cutting tube and an aluminum pedestal, also cooled with liquid nitrogen, located under the chamber. The frozen leaf discs were stored in liquid nitrogen before being assayed.