Ribulose-l,5-bisphosphate Carboxylase

Ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase) catalyzes the carboxylation of ribulose bisphosphate to give 3-phosphoglyceric acid [Eq. (14)] (7/. 72). 

RuBP carboxylase is the central C02-fixing enzyme in all plants thus, it is the most abundant enzyme in the biosphere (71, 72). The enzyme requires a divalent metal ion but has no other cofactors. CO2 is the substrate, rather than HCOs . 

Practically every kind of approach in enzymology has been applied to this enzyme, including kinetics, chemical modification. X-ray crystallography, site-directed mutagenesis, and isotope effects (7/, 72). Recent studies by X-ray crystallography (73-78) will particularly occupy our attention, as this is the only carboxylase for which an X-ray crystal structure has been reported. 

RuBP carboxylase has a number of unusual features. First, the enzyme must be activated by reaction with CO2 and metal prior to catalysis. Activation involves reaction of CO2 with an e-amino group of a lysine residue (Lys-191 in the enzyme from Rhodospirillum rubrum, Lys-201 in that from spinach) to form a carbamate that is then coordinated to the metal (75). This bound metal then forms part of the substrate-binding site. 

Second, the enzyme also catalyzes the oxygenation of ribulose bisphosphate, a reaction in which O2 serves as an alternate substrate and is competitive against CO2 [Eq. (15)]. 

---

Knight, S., Andersson, I., Branden, C.-I. Crystallographic analysis of ribulose-l,5-bisphosphate carboxylase from spinach at 2.4 A resolution. Subunit interactions and active site. /. Mol. Biol. 215 113-160,... 

Three-dimensional structure of ribulose-l,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum at 2.9 A resolution. EMBO J. 5, 3409-3415. 

Fmax at light saturation and at the optimal temperature for photosynthesis varies with plant species but is usually from 2 to 10 mol m-3 s-1. We can also estimate Vmax from measurements of the maximum rates of CO2 fixation by isolated chloroplasts. These maximum rates—which are sustained for short periods and are for optimal conditions—can be 100 mmol of CO2 fixed (kg chlorophyll)-1 s-1 [360 pmol (mg chlorophyll)-1 hour-1 in another common unit], which is approximately 3 mol m-3 s-1 (1 kg chlorophyll is contained in about 0.035 m3 of chloroplasts in vivo). In vitro, the key enzyme for CO2 fixation, ribulose-l,5-bisphosphate carboxylase/oxygenase, can have rates equivalent to 200 mmol (kg chlorophyll)-1 s-1. The estimates of Vmax using isolated chloroplasts or enzymes usually are somewhat lower than its values determined for a leaf Measurements using leaves generally indicate that KqOz is 5 to 20 mmol m-3. For instance, Kcch can be 9 mmol m-3 at 25°C with a Q10 of 1.8 (Woodrow and Berry, 1988 Q10 is defined in Chapter 3, Section 3.3B). 

Figure 8-13. Schematic illustration of Rubisco (ribulose-l,5-bisphosphate carboxylase/oxygenase) acting as the branch point for photosynthesis and photorespiration. All three of the organelles involved, but only a few of the biochemical steps, are indicated. ( represents phosphate. Note that 3-phosphoglycerate and glycolate refer to the dissociated forms of 3-phosphoglyceric acid and glycolic acid, respectively.)...

Corredor, J. E., Wawrik, B., Paul, J. H., Tran, H., Kerkhof, L., Lopez, J. M., et al. (2004). Geochemical rate-RNA integrated study Ribulose-l,5-bisphosphate carboxylase/oxygenase gene transcription and photosynthetic capacity of planktonic photoautotrophs. Appl. Environ. Microbiol. 70, 5459—5468. 

Ekman, P., Lignel, A., and Pedersen, M. (1989). Localization of ribulose-l,5-bisphosphate carboxylase/ oxygenase in Gracilaria secundata (Rhodophyta) and its role as a nitrogen storage pool. Bot. Mar. 32, 527-534. 

Wyman, M. (1999). Diel rhythms in ribulose-l,5-bisphosphate carboxylase/oxygenase and glutamine synthetase gene expression in a natural population of marine picoplanktonic cyanobacteria Syne-chococais spp.). Appl. Environ. Microbiol. 65, 3651—3659. 

Beudeker R. F., Cannon G. C., Kuenen J. G., and Shively J. M. (1980) Relations between D-ribulose-l,5-bisphosphate carboxylase, carboxysomes, and C02-fixing capacity in the obligately chemolithotroph Thiobacillus neapolitanus grown under different limitations in the chemostat. Arch. Microbiol 124, 185-189. 

It has been suggested that in certain parts of the oceans where dissolved zinc levels are extremely low, the limited availability of Zn might also limit CO2 fixation. This is because the Mg -dependent enzyme which fixes CO2, RuBisCO (ribulose-l,5-bisphosphate carboxylase/oxygenase), is a notoriously inefficient enzyme, with a low affinity for its substrate, CO2. It therefore requires high concentrations of CO2, and while cyanobacteria have evolved extremely effective mechanisms for uptake of CO2, which is then converted to HCOj, they need to regenerate CO2 from HCOj" in the vicinity of RuBisCO, and this requires the Zn -dependent enzyme, carbonic anhydrase. This was the first Zn -dependent enzyme to be discovered (Chapter 12) and virtually all carbonic anhydrases discovered to date have a catalytic Zn ion, bound to one His and two Cys residues. 

Fig.2.7 Assimilation of carbon dioxide. The key reaction of the dark reaction is the assimilation of carbon dioxide by ribulose l 5-bisphosphate carboxylase (Simplified from Fig. 26-31 in Biochemistry. L. Stryer, 4th Ed. 1995. W.H. Freeman Co., New York)...

Because plants must adapt to a wide variety of environmental conditions, the regulation of photosynthesis is complex. Although the control of most photosynthetic processes is far from being completely understood, several control features are well established. Most of these processes are directly or indirectly controlled by light. After a brief description of general light-related effects, the control of the activity of ribulose-l,5-bisphosphate carboxylase, the key regulatory enzyme in photosynthesis, is discussed. 

The increase in 02 and concomitant decrease in C02 caused by the evolution of oxygenic autotrophs on the Earth, has resulted in an undersaturation of the main (andancient) carboxylating enzyme, ribulose-l,5-bisphosphate carboxylase, responsible for the first step in carbon fixation—the dark reaction of photosynthesis. To palliate this difficulty, a number of species of marine phytoplankton have evolved carbon concentrating mechanisms that all involve some forms of... 

For the moment, we will focus on photoautotrophs that utilize rubisco. Rubisco is the official name of an enzyme for which the systematic name—ribulose-l,5-bisphosphate carboxylase oxygenase—is inconveniently long. As indicated by the first activity specified in the systematic name, this enzyme catalyzes the carboxylation of ribulose-1,5-bisphosphate, RuBP, a five-carbon molecule. A six-carbon product is formed as a transient intermediate, but the first stable products are two molecules of PGA, 3-phosphoglyceric acid, C3H7O7P. The carbon number of this compound gives the process its shorthand name, C3 photosynthesis. At physiological pH, the acidic functional groups on the reactants and products are ionized as shown below. 

The dark reactions of photosynthesis are actually stimulated by the light reactions. The central enzyme in the dark reactions, ribulose-l,5-bisphosphate carboxylase, is stimulated by high pH and C02 and... 

Ribulose-l,5-bisphosphate carboxylase (rubisco) is the central enzyme of the Calvin Cycle because it catalyzes the initial reaction is stage I, in which C02 is incorporated into ribulose-l,5-bisphosphate. This carboxylase activity occurs under conditions of low 02 and high C02. 

Quick, W.P., K. Fichtner, E.D. Schulze, R. Wendler, R.C. Leegood, H. Mooney, S.R. Rodermel, L. Bogorad, and M. Stitt Decreased ribulose-l,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed... 

Curmi, P.M.G., Cascio, D., Sweet, R.M., Eisenberg, D. and Schreuder, H. (1992) Crystal stmcture of the unactivated form of ribulose-l,5-bisphosphate carboxylase/oxygenase from tobacco refined at 2.0 A resolution. / Biol. Chem. 267 16980-16989. 

---