2-carboxyarabinitol 1-phosphate Research Articles

Non-canonical metabolic pathways in the malaria parasite detected by isotope-tracing metabolomics.

The malaria parasite, Plasmodium falciparum, proliferates rapidly in human erythrocytes by actively scavenging multiple carbon sources and essential nutrients from its host cell. However, a global overview of the metabolic capacity of intraerythrocytic stages is missing. Using multiplex 13C‐labelling coupled with untargeted mass spectrometry and unsupervised isotopologue grouping, we have generated a draft metabolome of P. falciparum and its host erythrocyte consisting of 911 and 577 metabolites, respectively, corresponding to 41% of metabolites and over 70% of the metabolic reaction predicted from the parasite genome. An additional 89 metabolites and 92 reactions were identified that were not predicted from genomic reconstructions, with the largest group being associated with metabolite damage‐repair systems. Validation of the draft metabolome revealed four previously uncharacterised enzymes which impact isoprenoid biosynthesis, lipid homeostasis and mitochondrial metabolism and are necessary for parasite development and proliferation. This study defines the metabolic fate of multiple carbon sources in P. falciparum, and highlights the activity of metabolite repair pathways in these rapidly growing parasite stages, opening new avenues for drug discovery.

Elucidating the biosynthesis of 2-carboxyarabinitol 1-phosphate through reduced expression of chloroplastic fructose 1,6-bisphosphate phosphatase and radiotracer studies with 14CO2.

2-carboxyarabinitol 1-phosphate limits photosynthetic CO2 assimilation at low light because it is a potent, naturally occurring inhibitor of ribulose 1,5-bisphosphate carboxylase/oxygenase. Evidence is presented that this inhibitor is derived from chloroplastic fructose 1,6-bisphosphate. First, transgenic plants containing decreased amounts of chloroplastic fructose 1,6-bisphosphate phosphatase contained increased amounts of fructose 1,6-bisphosphate and 2-carboxyarabinitol 1-phosphate and greatly increased amounts of the putative intermediates hamamelose and 2-carboxyarabinitol, which in some cases were as abundant as sucrose. Second, French bean leaves in the light were shown to incorporate 14C from 14CO2 sequentially into fructose 1,6-bisphosphate, hamamelose bisphosphate, hamamelose monophosphate, hamamelose, and 2-carboxyarabinitol. As shown previously, 14C assimilated by photosynthesis was also incorporated into 2-carboxyarabinitol 1-phosphate during subsequent darkness.

The discovery of Rubisco activase - yet another story of serendipity.

A brief history of Rubisco (ribulose bisphosphate carboxylase oxygenase) research and the events leading to the discovery and initial characterization of Rubisco activase are described. Key to the discovery was the chance isolation of a novel Arabidopsis photosynthesis mutant. The characteristics of the mutant suggested that activation of Rubisco was not a spontaneous process in vivo, but involved a heritable factor. The search for the putative factor by 2D electrophoresis identified two polypeptides, genetically linked to Rubisco activation, that were missing in chloroplasts from the mutant. An assay for the activity of these polypeptides, which were given the name Rubisco activase, was developed after realizing the importance of including ribulose bisphosphate (RuBP) in the assay. The requirement for ATP and the subsequent identification of activase as an ATPase came about fortuitously, the result of a RuBP preparation that was contaminated with adenine nucleotides. Finally, the ability of activase to relieve inhibition of the endogenous Rubisco inhibitor, 2-carboxyarabinitol 1-phosphate, provided an early indication of the mechanism by which activase regulates Rubisco.

Inactivation of the monocistronic rca gene in Anabaena variabilis suggests a physiological ribulose bisphosphate carboxylase/oxygenase activase-like function in heterocystous cyanobacteria.

There was no discernible effect after incubating recombinant Anabaena Rubisco and carboxyarabinitol 1-phosphate with the product of the Anabaena rca gene. Since the unactivated cyanobacterial Rubisco is not readily inhibited by ribulose 1,5-bisphosphate and fallover is not observed, a genetic basis for the function of the Rubisco activase-like gene (rca) was sought. The monocistronic rca gene was inactivated in vivo and resulting mutant strains of A. variabilis were found to be incapable of synthesizing immunologically detected RCA protein. The requirement for the product of the rca gene in the light was further examined by measuring Rubisco activity in permeabilized whole cells of wild-type and rca mutant strains at different light intensities. In a 1% CO2-air atmosphere, inactivation of rca reduced the ability of A. variabilis to elevate Rubisco activity under high light (73 micromol quanta m(-2) s(-1)), but had little effect under low light (8 micromol m(-2) s(-1)). For air-grown cultures, differences in the rates exhibited by the wild-type and rca mutant to fully activate Rubisco during a whole-cell assay were enhanced by increases in light intensity. The significance of the rca mutation was underlined by effects on growth as, unlike the wild-type, growth rates did not increase after cells transferred from low to high light intensities. Higher exogenous CO2 concentrations (1%) were required to sustain a normal growth rate for the A. variabilis rca mutant. When grown in air levels of CO2, the rca mutant not only needed longer times to double in cell density but also exhibited greatly diminished Rubisco activity compared with the wild-type strain. Despite the unusual properties of cyanobacterial Rubisco, these results suggest a physiological role for the product of the rca gene in maximizing the activity of Rubisco in heterocystous cyanobacteria.

Regulation of ribulose-1,5-bisphosphate Carboxylase/Oxygenase by carbamylation and 2-carboxyarabinitol 1-phosphate in tobacco: insights from studies of antisense plants containing reduced amounts of rubisco activase

The regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity by 2-carboxyarabinitol 1-phosphate (CA1P) was investigated using gas-exchange analysis of antisense tobacco (Nicotiana tabacum) plants containing reduced levels of Rubisco activase. When an increase in light flux from darkness to 1200 &mgr;mol quanta m-2 s-1 was followed, the slow increase in CO2 assimilation by antisense leaves contained two phases: one represented the activation of the noncarbamylated form of Rubisco, which was described previously, and the other represented the activation of the CA1P-inhibited form of Rubisco. We present evidence supporting this conclusion, including the observation that this second phase, like CA1P, is only present following darkness or very low light flux. In addition, the second phase of CO2 assimilation was correlated with leaf CA1P content. When this novel phase was resolved from the CO2 assimilation trace, most of it was found to have kinetics similar to the activation of the noncarbamylated form of Rubisco. Additionally, kinetics of the novel phase indicated that the activation of the CA1P-inhibited form of Rubisco proceeds faster than the degradation of CA1P by CA1P phosphatase. These results may be significant with respect to current models of the regulation of Rubisco activity by Rubisco activase.

Carboxyarabinitol 1-Phosphate Phosphatase from Leaves of Phaseolus vulgaris and Other Species

Carboxyarabinitol 1-Phosphate Phosphatase from Leaves of Phaseolus vulgaris and Other Species

Different location in dark-adapted leaves of Phaseolus vulgaris of ribulose-1,5-bisphosphate carboxylase/oxygenase and 2-carboxyarabinitol 1-phosphate

In situ RuBisCO activity was analyzed in order to determine whether 2-carboxyarabinitol 1-phosphate (CA1P) interacts with the enzyme in dark-adapted leaves of Phaseolus vulgaris. Leaves ground to fine powder in liquid nitrogen were put directly into a reaction mixture containing a saturating concentration of ribulose 1,5-bisphosphate (RuBP) to preserve the activity of RuBisCO which was in the chloroplasts. Some 70% of the total catalytic sites of RuBisCO possessed carboxylase activity in this assay, however, RuBisCO was inhibited in the absence of RuBP. CA1P seemed to be concentrated in the veins. These results indicate that RuBisCO was not complexed with CA1P in leaves.

2-Carboxyarabinitol 1-phosphate (CA1P) formation through a phosphate exchange reaction catalysed by the CA1P phosphatase from French bean (Phaseolus vulgaris L.).

Transfer of the phosphate group of 2-carboxy-D-arabinitol 1-phosphate (CA1P) to 14C-labelled 2-carboxy-D-arabinitol (CA) was catalysed by extracts from leaves of Phaseolus vulgaris. This phosphotransferase activity co-purified with CA1P phosphatase, described previously. This activity was increased, up to 16-fold, by addition of bicarbonate ions at pH 9-10, suggesting rate enhancement by enzyme carbamylation. A V(max) of 1.5 mumol/min per mg of protein and a K(m) (for CA) of 1.8 mM were estimated for the exchange reaction, with the purified phosphatase. 2-Carboxy-D-arabinitol 1,5-bisphosphate and 2-carboxy-D-ribitol 1,5-bisphosphate, but not D-ribulose 1,5-bisphosphate, could replace CA1P as phosphate donor to [14C]CA.

Levels of Carboxyarabinitol 1-Phosphate and Related Photosynthetic Components during Leaf Development in Phaseolus vulgaris

Levels of Carboxyarabinitol 1-Phosphate and Related Photosynthetic Components during Leaf Development in Phaseolus vulgaris

The absence of Rubisco activase activity in total wheat leaf extracts is recovered in the purified protein

When desalted extracts of soluble protein from dark-adapted wheat leaves were assayed for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase activity in the presence of 1 mM ATP and an ATP-regenerating system, very little ATP-dependent activation of RuBP-inactivated Rubisco was found. In extracts from light-adapted leaves a very similar pattern of Rubisco activation was observed except that the overall level of Rubisco activity was much lower than in the extracts from dark-adapted leaves. These features were apparent both at low (120 μg per ml) and high (640 μg per ml) protein concentrations. We were unable to demonstrate Rubisco activase activity in crude leaf extracts. Consequently, in order to establish that Rubisco activase was present in wheat leaf extracts the wheat leaf protein was purified to homogeneity. The identity of the protein was confirmed with antibodies to the spinach enzyme, ATPase activity and activase-mediated release of the inhibitor, carboxyarabinitol-1-phosphate (CA1P) from the tertiary Rubisco complex. The pure wheat Rubisco activase relieved the CA1P-induced inhibition of Rubisco activity. Rubisco activase had no significant effect on the affinity of wheat Rubisco for the substrate, ribulose-1,5-bisphosphate (RuBP).

Evidence That 2-Carboxyarabinitol 1-Phosphate Binds to Ribulose-1,5-Bisphosphate Carboxylase in Vivo.

An important question concerning the role of carboxyarabinitol 1-phosphate (CA1P) metabolism in the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity is the extent to which CA1P is bound to Rubisco in vivo. We report here the development of an extraction procedure using ammonium sulfate that stabilizes CA1P bound to Rubisco. This procedure exploits the ability of sulfate to bind at the catalytic site of Rubisco and to competitively balance the binding and release of CA1P from Rubisco. In darkened bean leaves about 75% of the Rubisco catalytic sites were found to be bound with CA1P. This confirms previous indirect estimates from gas exchange measurements. We have used this extraction procedure to examine CA1P-Rubisco interactions in bean during a natural transition from darkness to light. With increasing light intensity following sunrise, CA1P degradation proceeded in two distinct phases: first, a majority of the unbound CA1P pool was degraded at very low light levels ([less than or equal to]30 [mu]mol quanta m-2 s-1); second, CA1P initially bound to Rubisco was then degraded at increasing light levels (>30 [mu]mol quanta m-2 s-1). These results indicate that there is a low-fluence activation of CA1P phosphatase that can occur prior to CA1P release by Rubisco activase. This activation may be mediated by NADPH. During sunrise in bean, the level of the catalytically competent form of Rubisco was regulated by CA1P metabolism.

Intraspecific variation in the light/dark modulation of ribulose 1,5-bisphosphate carboxylase-oxygenase activity in soybean

A comparative study was made of the inhibition of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) amongst six cultivars of Glycine max L. Merr., associated with synthesis of 2-carboxyarabinitol 1-phosphate (CA1P) during darkness. Significantly lower mean values of dark inhibition of Rubisco were observed in soybean cv. Davis than in cvs Bragg, Cobb, Hardee, Gordon, and Kirby. The CA1P synthesis/degradation cycle during dark/light transitions remained operational in cv. Bragg plants grown at low irradiance (40 μmol photons m -2 s -1 )

Long-term kinetics of the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in plants with and without 2-carboxyarabinitol 1-phosphate

The light-dependent modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity was studied in two species: Phaseolus vulgaris L., which has high levels of the inhibitor of Rubisco activity, carboxyarabinitol 1-phosphate (CA1P), in the dark, and Chenopodium album L., which has little CA1P. In both species, the ratio of initial to fully-activated Rubisco activity declined by 40–50% within 60 min of a reduction in light from high a photosynthetic photon flux density (PPFD; >700 μmol · m−2 · s−1) to a low PPFD (65 ± 15 μmol · m−2 · s−1) or to darkness, indicating that decarbamylation of Rubisco is substantially involved in the initial regulatory response of Rubisco to a reduction in PPFD, even in species with potentially extensive CA1P inhibition. Total Rubisco activity was unaffected by PPFD in C. album, and prolonged exposure (2–6 h) to low light or darkness was accompanied by a slow decline in the activity ratio of this species. This indicates that the carbamylation state of Rubisco from C. album gradually declines for hours after the large initial drop in the first 60 min following light reduction. In P. vulgaris, the total activity of Rubisco declined by 10–30% within 1 h after a reduction in PPFD to below 100 μmol · m−2 · s−1, indicating CA1P-binding contributes significantly to the reduction of Rubisco capacity during this period, but to a lesser extent than decarbamylation. With continued exposure of P. vulgaris leaves to very low PPFDs (< 30 μmol · m−2 · s−1), the total activity of Rubisco declined steadily so that after 6–6.5 h of exposure to very low light or darkness, it was only 10–20% of the high-light value. These results indicate that while decarbamylation is more prominent in the initial regulatory response of Rubisco to a reduction in PPFD in P. vulgaris, binding of CA1P increases over time and after a few hours dominates the regulation of Rubisco activity in darkness and at very low PPFDs.

Regulation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activity in Response to Reduced Light Intensity in C4 Plants.

The light-dependent regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity was studied in 16 species of C4 plants representing all three biochemical subtypes and a variety of taxonomic groups. Rubisco regulation was assessed by measuring (a) the ratio of initial to total Rubisco activity, which reflects primarily the carbamylation state of the enzyme, and (b) total Rubisco activity per mol of Rubisco catalytic sites, which declines when 2-carboxyarabinitol 1-phosphate (CA1P) binds to carbamylated Rubisco. In all species examined, the activity ratio of Rubisco declined with a reduction in light intensity, although substantial variation was apparent between species in the degree of Rubisco deactivation. No relationship existed between the degree of Rubisco deactivation and C4 subtype. Dicots generally deactivated Rubisco to a greater degree than monocots. The total activity of Rubisco per catalytic site was generally independent of light intensity, indicating that CA1P and other inhibitors are not major contributors to the light-dependent regulation of Rubisco activity in C4 plants. The light response of the activity ratio of Rubisco was measured in detail in Amaranthus retroflexus, Brachiaria texana, and Zea mays. In A. retroflexus and B. texana, the activity ratio declined dramatically below a light intensity of 400 to 500 [mu]mol of photons m-2 s-1. In Z. mays, the activity ratio of Rubisco was relatively insensitive to light intensity compared with the other species. In A. retroflexus, the pool size of ribulose bisphosphate (RuBP) declined with reduced light intensity except between 50 and 500 [mu]mol m-2 s-1, when the activity ratio of Rubisco was light dependent. In Z. mays, by contrast, the pool size of RuBP was light dependent only below 350 [mu]mol m-2 s-1. These results indicate that, in response to changes in light intensity, most C4 species regulate Rubisco by reversible carbamylation of catalytic sites, as commonly observed in C3 plants. In a few species, notably Z. mays, Rubisco is not extensively regulated in response to changes in light intensity, possibly because the activity of the CO2 pump may become limiting for photosynthesis at subsaturating light intensity.

Light-dependent modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in the genus Phaseolus

Modulation of the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in low light and darkness was measured in A) 25 genotypes from the four cultivated species of Phaseolus (P. vulgaris, P. acutifolius, P. lunatus and P. coccineus), B) 8 non-cultivated Phaseolus species, and C) the related species Macroptileum atropurpureum. The activity ratio of Rubisco (the ratio of initial and total Rubisco activities, which reflects Rubisco carbamylation), and the molar activity of fully-activated Rubisco (which primarily reflects the inhibition of Rubisco activity by carboxyarabinitol 1-phosphate, CA1P) were assayed in leaves from the cultivated species sampled at midday in full sunlight, in low light at dusk (60 to 100 μmol photons m(-2)s(-1)), and after at least 4 h in darkness. Dark inhibition of Rubisco molar activity was compared in both cultivated and non-cultivated species. In all cultivated genotypes, a significant reduction of the activity ratio of Rubisco was measured in leaves sampled at low light; however, the molar activity of fully activated Rubisco was not greatly reduced in these low light samples. In darkened leaves, molar activities substantially declined in most Phaseolus species with 11 of 13 exhibiting greater than 60% reduction. In P. vulgaris, the reduction of molar activity was extensive (greater than 69%) in all genotypes studied, which included wild progenitors as well as ancient and advanced cultivars. These results indicate that at low light late in the day, modulation of Rubisco activity is primarily through changes in carbamylation state, with CA1P playing a more limited role. By contrast in the dark, binding of CA1P dominates the modulation of Rubisco activity in Phaseolus in a pattern that appears to be conserved within a species, but can vary significantly between species within a genus. The degree of CA1P inhibition in Phaseolus was associated with phylogenetic affinities within the genus, as the species with extensive dark-inhibition of Rubisco activity tended to be more closely related to each other than to species with reduced inhibition of Rubisco activity.

Subunit interactions of Rubisco activase: Polyethylene glycol promotes self-association, stimulates ATPase and activation activities, and enhances interactions with Rubisco

The effect of polyethylene glycol (PEG) on the enzymatic and physical properties of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase was examined. In the presence of PEG, Rubisco activase exhibited higher ATPase and Rubisco activating activities, concomitant with increased apparent affinity for ATP and Rubisco. Specific ATPase activity, which was dependent on Rubisco activase concentration, was also higher in the presence of Ficoll, polyvinylpyrrolidone, and bovine serum albumin. The ability of Rubisco activase to facilitate dissociation of the tight-binding inhibitor 2-carboxyarabinitol 1-phosphate from carbamylated Rubisco was also enhanced in the presence of PEG. Mixing experiments with Rubisco activase from two different sources showed that tobacco Rubisco activase, which exhibited little activation of spinach Rubisco by itself, was inhibitory when included with spinach Rubisco activase. Polyethylene glycol improved the ability of tobacco and a mixture of tobacco plus spinach Rubisco activase to activate spinach Rubisco. Estimates based on rate zonal sedimentation and gel-filtration chromatography indicated that the apparent molecular mass of Rubisco activase was two- to fourfold higher in the presence of PEG. The increase in apparent molecular mass was consistent with the propensity of solvent-excluding reagents like PEG to promote self-association of proteins. Likewise, the change in enzymatic properties of Rubisco activase in the presence of PEG and the dependence of specific activity on protein concentration resembled changes that often accompany self-association. For Rubisco activase, high concentrations of protein in the chloroplast stroma would provide an environment conducive to self-association and cause expression of properties that would enhance its ability to function efficiently in vivo.

Metabolism of 2′-Carboxyarabinitol in Leaves

Results presented here indicate that 2'-carboxyarabinitol (CA) is the in vivo precursor and product of 2'-carboxyarabinitol 1-phosphate (CA1P) metabolism in leaves. When [2-(14)C]CA was fed in the light to leaves of five species known to be highly active in CA1P metabolism (Phaseolus vulgaris, Lycopersicon esculentum, Helianthus annuus, Petunia hybrida, and Beta vulgaris), [(14)C]CA1P was formed in the dark. Reillumination of a Phaseolus leaf caused this [(14)C]CA1P to be rapidly metabolized to [(14)C]CA (t((1/2)) = 1 min). The epimer 2'-carboxyribitol could not substitute for CA in the dark synthesis of CA1P, and CA in the anionic form was a better substrate than CA in the lactone form. In leaves of Phaseolus vulgaris, the active CA pool size used in the dark synthesis of CA1P is between about 70 and 110 nanomoles per milligram of chlorophyll. The photosynthetic electron transport inhibitor diuron did not affect the dark synthesis of [(14)C]CA1P, but did greatly reduce the rate of its subsequent light degradation (t((1/2)) = approximately 10 min). Dark synthesis of [(14)C]CA1P was inhibited by dithiothreitol and NaF. From the present data, we suggest that CA1P and CA participate in a metabolic substrate cycle in vivo.

Identification and Levels of 2′-Carboxyarabinitol in Leaves

2'-Carboxyarabinitol 1-phosphate (CA1P) is a naturally occurring inhibitor of ribulose-1,5 bisphosphate carboxylase/oxygenase activity. A chloroplast phosphatase has previously been identified that degrades CA1P in vitro to carboxyarabinitol (CA) plus phosphate, but CA has not yet been detected in plants. Here, we detail procedures to isolate and assay CA from leaves and utilize mass spectrometry to demonstrate for the first time that CA is present in plants. CA was present in leaves of all 13 species examined, including those of C(3), C(4), and Crassulacean acid metabolism photosynthetic subgroups. CA was present both in species with high levels of CA1P (e.g. Phaseolus vulgaris, Lycopersicon esculentum, Beta vulgaris) as well as in species with low levels of CA1P (e.g. Spinacea oleracea, Triticum aestivum). CA levels in the light were sometimes greater than those in the dark. Bean leaves had the most CA of any species tested, with levels in the light approaching 1 micromole per milligram of chlorophyll. In illuminated bean leaves, about 63% of the CA is located outside the chloroplast. CA is one of only a few branched chain sugar acids to be identified from plants.

Dark inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase in legumes: A biosystematic study

2-carboxyarabinitol 1-phosphate (CA 1-P) is a naturally occurring inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Members of the Fabaceae exhibit a particularly wide range in the extent of CA 1-P accumulation during darkness and include Phaseolus vulgaris, whose dark/light regulation of Rubisco activity is principally achieved by synthesis/degradation of CA 1-P. An extensive survey of the degree of dark inhibition of Rubisco was undertaken for the subfamily Papilionoideae to elucidate evolutionary patterns in the occurrence of this regulatory mechanism. Seventy-five species from 21 tribes were examined. Dark inhibition of Rubisco was found in ancestral tribes such as the Sophoreae, but was substantially reduced or absent in representative species of three more recently evolved tribes, Cicereae, Hedysareae and Vicieae. We conclude that regulation of Rubisco by CA 1-P is neither of recent origin nor of restricted distribution among the Papilionoideae. On the contrary, it becomes lost or less pronounced only in a minority of the more evolutionarily advanced species in this important subfamily.

Xylulose 1,5-Bisphosphate Synthesized by Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase during Catalysis Binds to Decarbamylated Enzyme

Xylulose 1,5-bisphosphate (XuBP) is synthesized from ribulose 1,5-bisphosphate (RuBP) at carbamylated catalytic sites on ribulose 1,5-bisphosphate carboxylase (Rubisco) with significant amounts of XuBP being formed at pH less than 8.0. XuBP has been separated by high performance liquid chromatography and identified by pulsed amperometry from compounds bound to Rubisco during catalysis with the purified enzyme and from celery (Apium graveolens var Utah) leaf extracts. XuBP does not bind tightly to carbamylated sites, but does bind tightly to decarbamylated sites. Upon incubation of fully activated Rubisco with 5 micromolar XuBP, loss of activator CO(2) occurred before XuBP bound to the enzyme catalytic sites, even in the presence of excess CO(2) and Mg(2+). Binding of XuBP to decarbamylated Rubisco sites was highly pH dependent. At pH 7.0 and 7.5 with 10 millimolar MgCl(2) and 10 millimolar KHCO(3), the apparent dissociation constant for XuBP, K(d), was 0.03 micromolar, whereas at pH 8.0 and 8.5, the apparent K(d) was 0.35 and 2.0 micromolar, respectively. This increase in K(d) with pH was a result of a decrease in the association rate constant and an increase in the dissociation rate constant of XuBP bound to decarbamylated sites on Rubisco. The K(d) of 2-carboxyarabinitol 1-phosphate binding to carbamylated sites was only slightly pH dependent.