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

TLDR

Rubisco, the CO₂‑fixing enzyme essential for autotrophy, is limited by slow catalysis and a confusing preference for CO₂ versus O₂, a longstanding puzzle. The authors propose that this limitation arises because CO₂ binding is difficult, forcing the enzyme to determine specificity largely in the transition state, and that natural selection has shaped a transition state resembling a carboxylate to enhance CO₂/O₂ discrimination. By maximizing structural differences between carboxylation and oxygenation transition states, the enzyme improves discrimination, but the resulting similarity to the carboxyketone product tightens binding and slows product release. The study concludes that Rubiscos are nearly perfectly optimized for their cellular CO₂/O₂ and thermal environments, explaining their modest catalytic enhancement, the link between CO₂/O₂ specificity and carbon isotope fractionation, and predicting reduced effectiveness at higher temperatures.

Abstract

The cornerstone of autotrophy, the CO(2)-fixing enzyme, d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is hamstrung by slow catalysis and confusion between CO(2) 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 CO(2) 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 CO(2)/O(2) specificity, in response to reducing atmospheric CO(2):O(2) ratios, has resulted in a transition state for CO(2) addition in which the CO(2) 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 CO(2), O(2), and thermal conditions in their subcellular environments, optimizing this compromise between CO(2)/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 CO(2)/O(2) specificity and (12)C/(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.