Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized

Abstract

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...

Adaptive signals in algal Rubisco reveal a history of ancient atmospheric carbon dioxide

“…This relaxes pressure for Rubisco to have a high affinity for CO 2 and therefore allows an increase in V c , which results in increased photosynthetic efficiency as the plant requires less nitrogen to achieve a given CO 2 [4,6,20] and K c (dark blue bars) [6,21] with standard error bars and number of species measured (n) are shown. [55,56].…”

“…Thermodynamic constraints dictate a trade-off between carboxylation velocity (V c ) and affinity for CO 2 that has led to suggestions that despite being conserved and sluggish, Rubisco is optimized to its physical environment [4]. The variation in Rubisco catalytic properties found within photosynthetic eukaryotes hints that it has undergone adaptations to low CO 2 [5][6][7][8], but little is known about the detailed timing of evolution of Rubisco and its relationship to the environmental change.…”

Constraining the timing of the Great Oxidation Event within the Rubisco phylogenetic tree

“…Rubisco also catalyzes a competing photorespiration reaction in which RuBP is combined with oxygen, which in turn reduces the overall metabolic efficiency of carbon fixation. This chemical competition is thought to derive from the relatively featureless structural attributes of CO 2 and O 2 , which force substrate specificity to be determined largely in the transition state catalyzed by the enzyme rather than at the initial point of substrate binding (Tcherkez, Farquhar, & Andrews, 2006). …”

Evolution of C 4 plants: a new hypothesis for an interaction of CO 2 and water relations mediated by plant hydraulics

“…This difference arises because the active site of Rubisco is unable to discriminate completely between CO 2 and O 2 , and catalyses the fixation of both molecules [23,24]. The oxygenation reaction generates toxic intermediates that must be metabolized via photorespiration to render them harmless and to recover carbon.…”