Subsaturating Ribulose-1,5-Bisphosphate Concentration Promotes Inactivation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) (Studies Using Continuous Substrate Addition in the Presence and Absence of Rubisco Activase)

Abstract

, lllinois 61801 (A.R.P.)We developed a continuous-addition method for maintaining subsaturating concentrations of ribulose-1,5-bisphosphate (RuBP) for severa1 minutes, while simultaneously monitoring its consumption by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This method enabled us to observe the effects of subsaturating RuBP and CO, concentrations on the activity of Rubisco during much longer periods than previously studied. At saturating CO,, the activity of the,enzyme declined faster when RuBP was maintained at concentrations near its K,,, value than when RuBP was saturating. At saturating RuBP, activity declined faster at limiting than at saturating CO,, i n accordance with previous observations. The most rapid decline i n activity occurred when both CO, and RuBP concentrations were subsaturating. The activity loss was accompanied by decarbamylation of the enzyme, even though the enzyme was maintained at the same CO, concentration before and after exposure to RuBP. Rubisco activase ameliorated the decline in activity at subsaturating CO, and RuBP concentrations. The results are consistent with a proposed mechanism for regulating the carbamylation of Rubisco, which postulates that Rubisco activase counteracts Rubisco's unfavorable carbamylation equilibrium i n the presence of RuBP by accelerating, in an ATP-dependent manner, the release of RuBP from its complex with uncarbamylated sites.During photosynthesis, the activity of Rubisco (EC 4.1.1.39) must be modulated to balance the rate of RuBP consumption by carboxylation and oxygenation with the rate of RuBP regeneration. Metabolic conditions in the chloroplast are subject to variations induced by environmental fluctuations (such as varying irradiance) or by developmental changes (such as varying sink strength). Extensive study of the regulation of Rubisco has shown that,The majority of this work was performed while A.R.P. was a Visiting Fellow at the Research School of Biological Sciences and followed initial studies using a 14C0, assay with R.M.L. during his earlier visit to the University of Illinois.* Corresponding author; e-mail arportis@uiuc.edu; fax 1-217-244-4419. 1441under steady-state conditions, the activity is regulated by variations in the activation state of the enzyme rather than by fluctuations in RuBP concentration in the subsaturating range (Portis, 1992;Geiger and Servaites, 1994). The activation state is varied by changing the carbamylation status of the enzyme (the fraction of active sites having carbamylated Lys at residue 201, which permits binding of the catalytically essential divalent metal). In a minority of plant species, the activity is also varied by the inhibitor, CAIP, which reduces activity by binding to ECM, particularly in darkness. As a consequence of this mode of Rubisco regulation, RuBP generally remains a t saturating levels in the stroma during illumination. RuBP also binds to E with a Kd of approximately 20 nM, forming a stable, inactive complex (Jordan and Chollet, 1983). The activation state of R...

Effect of heat stress on the inhibition and recovery of the ribulose-1,5-bisphosphate carboxylase/oxygenase activation state

“…Ribulose-1,5-bisphosphate is a potent inhibitor of decarbamylated Rubisco (Chu and Bassham 1975;Jordan and Chollet 1983), and this inhibition is ameliorated by Rubisco activase (Portis et al 1995).…”

Kinetic Analysis of the Slow Inactivation of Rubisco During Catalysis: Effects of Temperature, O2 and Mg++

“…The potential mechanism(s) responsible for Rubisco fallover are thought to be the loss of catalytically competent sites due to either the tight binding of inhibitory isomers of ribulose-P 2 and/or decarbamylation (Edmondson et al 1990c;Portis et al 1995;Zhu et al 1998). The inhibitors appear to be generated by isomerization and rearrangement of ribulose-P 2 in the active site during Rubisco catalysis (Paech et al 1978;Chen and Hartman 1995;Pearce and Andrews 2003).…”

“…Rubisco fallover under various conditions was initially characterized in several pioneering reports (Edmondson et al 1990a;Zhu and Jensen 1991b;Portis et al 1995). In these studies, low levels of ribulose-P 2 , CO 2 , and pH were reported to increase the extent and/or rate of fallover.…”

Specificity for Activase Is Changed by a Pro-89 to Arg Substitution in the Large Subunit of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase

“…Activase Specificity Assay-The ability of RuBP-inhibited Rubisco to be activated by Rubisco activase was assessed by a spectrophotometric assay at 25°C that couples the product of RuBP carboxylation, 3-phosphoglycerate, to the oxidation of NADH (10,39). Reaction mixtures contained 50 mM Tricine, pH 8.0, 12 mM MgCl 2 , 10 mM NaHCO 3 , 10 mM dithiothreitol, 2 mM ATP, 2 mM RuBP, 10 mM phosphocreatine, 750 M NADH, and coupling enzymes (60 IU/ml 3-phosphoglycerate kinase, 300 IU/ml triose-phosphate isomerase, and 30 IU/ml each of creatine phosphokinase, glyceraldehyde-3-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, and carbonic anhydrase).…”

“…Activase couples the energy of ATP hydrolysis to the release of inhibitory sugar phosphates bound to both carbamylated and decarbamylated Rubisco active sites. High activase activity, along with high concentrations of RuBP, allows rapid and complete carbamylation to occur via a quasi-equilibrium state (10,11) and reverses inhibition by misprotonated sugar phosphates (12). Activase is essential for photosynthesis in higher plants.…”