The cornerstone of autotrophy, the CO2-fixing enzyme, D-ribulose-1,5-bisphosphate carboxylase͞oxygenase (Rubisco), is hamstrung by slow catalysis and confusion between CO2 and O2 as substrates, an ''abominably perplexing'' puzzle, in Darwin's parlance. Here we argue that these characteristics stem from difficulty in binding the featureless CO2 molecule, which forces specificity for the gaseous substrate to be determined largely or completely in the transition state. We hypothesize that natural selection for greater CO2͞O2 specificity, in response to reducing atmospheric CO2:O2 ratios, has resulted in a transition state for CO2 addition in which the CO2 moiety closely resembles a carboxylate group. This maximizes the structural difference between the transition states for carboxylation and the competing oxygenation, allowing better differentiation between them. However, increasing structural similarity between the carboxylation transition state and its carboxyketone product exposes the carboxyketone to the strong binding required to stabilize the transition state and causes the carboxyketone intermediate to bind so tightly that its cleavage to products is slowed. We assert that all Rubiscos may be nearly perfectly adapted to the differing CO2, O2, and thermal conditions in their subcellular environments, optimizing this compromise between CO2͞O2 specificity and the maximum rate of catalytic turnover. Our hypothesis explains the feeble rate enhancement displayed by Rubisco in processing the exogenously supplied carboxyketone intermediate, compared with its nonenzymatic hydrolysis, and the positive correlation between CO2͞O2 specificity and 12 C͞ 13 C fractionation. It further predicts that, because a more product-like transition state is more ordered (decreased entropy), the effectiveness of this strategy will deteriorate with increasing temperature. enzyme mechanisms ͉ isotope fractionation ͉ transition states T he most abundant protein in nature is D-ribulose 1,5-bisphosphate (RuBP) carboxylase͞oxygenase (Rubisco, EC 4.1.1.39) (1). This immense N investment is required to counter the enzyme's pitifully sluggish catalytic performance. Furthermore, Rubisco's tendency to confuse the substrate of photosynthesis, CO 2 , with the product, O 2 , saddles all aerobic photosynthetic organisms with energy-wasting photorespiration (2). Thus, this single enzyme's efficiency, or lack thereof, dictates the (in)efficiency with which plants use their basic resources of light, water, and N, and currently intense biotechnological effort aims to improve its catalytic properties and to engineer such improvements into crop plants (3, 4). Rubisco's difficulties stem from the inevitable O 2 sensitivity of the 2,3-enediol form of RuBP, to which CO 2 is added during the carboxylase reaction (5), which causes carboxylase͞oxygenase bifunctionality (Fig. 1). This difficulty is exacerbated by the need to discriminate between featureless molecules, CO 2 and O 2 , that can be bound in Michaelis-Menten complexes only weakly, if at all (2, 6). R...

Ribulose-1,5-bisphosphate carboxylase was activated by incubation with CO2 and Mg2++, and inactivated upon removal of CO2 and Mg2+ by gel filtration. The activation process involved CO2 rather than HCO3-. The activity of the enzyme was dependent upon the preincubation concentrations of CO2 and Mg2+ and upon the preincubation pH, indicating that activation involved the reversible formation of an equilibrium complex of enzyme-CO2-Mg. The initial rate of activation was linearly dependent upon the CO2 concentration but independent of the Mg2+ concentration. Kinetic analyses indicated that the enzyme reacted first with CO2 in a rate-determining and reversible step, followed by a rapid reaction with Mg2+ to form an active ternary complex (see eq 1 in text). The pseudo-first order rate constant, kobsd, for the activation process at constant pH was derived: kobsd=k1[CO2] + (k2k4/k3[Mg2+]). Experimentally, kobsd was shown to be linearly dependent upon the CO2 concentration and inversely dependent upon the Mg2+ concentration. The activity of the enzyme after preincubation to equilibrium at constant concentrations of CO2 and Mg2+ increased as the preincubation pH was raised, indicating that CO2 reacted with an enzyme group whose pK was distinctly alkaline. It is proposed that the activation of ribulose-1, 5-biphosphate carboxylane involves the formation of a carbamate.

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In 27 C 4 grasses grown under adequate or deficient nitrogen (N) supplies, N-use efficiency at the photosynthetic (assimilation rate per unit leaf N) and whole-plant (dry mass per total leaf N) level was greater in NADP-malic enzyme (ME) than NAD-ME species. This was due to lower N content in NADP-ME than NAD-ME leaves because neither assimilation rates nor plant dry mass differed significantly between the two C 4 subtypes. Relative to NAD-ME, NADP-ME leaves had greater in vivo (assimilation rate per Rubisco catalytic sites) and in vitro Rubisco turnover rates (k cat ; 3.8 versus 5.7 s 21 at 25°C). The two parameters were linearly related. In 2 NAD-ME (Panicum miliaceum and Panicum coloratum) and 2 NADP-ME (Sorghum bicolor and Cenchrus ciliaris) grasses, 30% of leaf N was allocated to thylakoids and 5% to 9% to amino acids and nitrate. Soluble protein represented a smaller fraction of leaf N in NADP-ME (41%) than in NAD-ME (53%) leaves, of which Rubisco accounted for one-seventh. Soluble protein averaged 7 and 10 g (mmol chlorophyll) 21 in NADP-ME and NAD-ME leaves, respectively. The majority (65%) of leaf N and chlorophyll was found in the mesophyll of NADP-ME and bundle sheath of NAD-ME leaves. The mesophyll-bundle sheath distribution of functional thylakoid complexes (photosystems I and II and cytochrome f ) varied among species, with a tendency to be mostly located in the mesophyll. In conclusion, superior N-use efficiency of NADP-ME relative to NAD-ME grasses was achieved with less leaf N, soluble protein, and Rubisco having a faster k cat .C 4 photosynthesis involves the close collaboration of two photosynthetic cell types, the mesophyll (M) and bundle sheath (BS). A key characteristic of the C 4 syndrome is the operation of a CO 2 concentrating mechanism, which serves to raise the CO 2 concentration in the BS around Rubisco to levels high enough to suppress photorespiration and almost saturate photosynthesis in air (Hatch, 1987). This explains the commonly observed high photosynthetic rates of C 4 relative to C 3 leaves, when comparisons are made under high light and temperature. C 4 plants also have greater photosynthetic rates and accumulate more biomass than C 3 plants for less leaf nitrogen (N) and Rubisco (Bolton and Brown, 1978;Brown, 1978;Schmitt and Edwards, 1981;Ghannoum et al., 1997;Ghannoum and Conroy, 1998;Makino et al., 2003). The C 4 photosynthetic pathway is divided into three biochemical subtypes following the major C 4 acid decarboxylation enzyme (NAD-malic enzyme [ME], NADP-ME, and phosphoenolpyruvate carboxykinase; Hatch, 1987). C 4 grasses with different biochemical subtypes have characteristic leaf anatomy (Hattersley, 1992) and different geographic distribution according to rainfall, such as seen in Australia (Hattersley, 1992) and South Africa (Ellis et al., 1980). With increasing rainfall, NADP-ME grasses increase in abundance, whereas NAD-ME grasses become less abundant. The aforementioned observations triggered our interest in the comparative physiology of the C 4 subtypes, espec...

The marine cyanobacterium, Synechococcus sp. Nageli (strain RRIMP N1) changes its affinity for external inorganic carbon used in photosynthesis, depending on the concentration of CO2 provided during growth. The high affinity for CO2 + HCO-of air-grown cells (K1/2 < 80 nanomoles [pH 8.21) would seem to be the result of the presence of an inducible mechanism which concentrates inorganic carbon (and thus C02) within the cells. Silicone-oil centrifugation experiments indicate that the inorganic carbon concentration inside suitably induced cells may be in excess of 1,000-fold greater than that in the surrounding medium, and that this accumulation is dependent upon light energy. The quantum requirements for 02 evolution appear to be some 2-fold greater for low CO0-grown cells, compared with high C02-grown cells. This presumably is due to the diversion of greater amounts of light energy into inorganic carbon transport in these cells.A number of experimental approaches to the question of whether CO2 or HCO3-is primarily utilized by the inorganic carbon transport system in these cells show that in fact both species are capable of acting as substrate. C02, however, is more readily taken up when provided at an equivalent concentration to HCO3-.This discovery suggests that the mechanistic basis for the inorganic carbon concentrating system may not be a simple HC03-pump as has been suggested. It is clear, however, that during steady-state photosynthesis in seawater equilibrated with air, HC03-uptake into the cell is the primary source of internal inorganic carbon.The utilization of inorganic carbon by cyanobacteria has recently been the subject of several investigations (6, 9). It is clear that these photosynthetic organisms are able to change their relative affinities for external inorganic carbon, depending on the level in the external medium, and that there is some active accumulation mechanism which allows CO2 to be concentrated inside the cell. The concentrating mechanism has been proposed to be an HCO3 pump (3), rather than a CO2 utilizing system, which would function most efficiently at alkaline pH where a large portion of the inorganic carbon is present as HCO3 . An environment where a HCO3 transport mechanism could be expected to play a major role in the fixation of carbon from the aqueous medium is seawater, where a more or less constant alkaline pH of around 8.2 exists, providing a constant high level of HCO3 . Experiments described here were aimed at characterizing the photosynthetic properties of a marine cyanobacterium, Synechococcus sp., and assessing to what extent HCO3 rather than CO2 is used for photosynthesis.We have used a combination of techniques, including silicone oil centrifugation and mass spectrometric monitoring of gas exchange, to show that this cyanobacterium does possess an inducible active CO2 concentrating system. In exploring the question of active species used by this concentrating mechanism, we are forced to conclude that both CO2 and HC03-can both act as substrates; however, durin...