The cornerstone of autotrophy, the CO2-fixing enzyme, (D)-ribulose-,5-bisphosphate carboxylase/oxygenase (Rubisco), is hamstrung by slow catalysis and confusion between CO2 and O-2 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 CO2/O-2 specificity, in response to reducing atmospheric CO2:O-2 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, O-2, and thermal conditions in their subcellular environments, optimizing this compromise between CO2/O-2 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 CO2/O-2 specificity and C-12/C-13 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.
