Abstract
Interprotein and enzyme-substrate couplings in interfacial biocatalysis induce spatial correlations beyond the capabilities of classical mass-action principles in modeling reaction kinetics. To understand the impact of spatial constraints on enzyme kinetics, we developed a computational scheme to simulate the reaction network of enzymes with the structures of individual proteins and substrate molecules explicitly resolved in the three-dimensional space. This methodology was applied to elucidate the rate-limiting mechanisms of crystalline cellulose decomposition by cellobiohydrolases. We illustrate that the primary bottlenecks are slow complexation of glucan chains into the enzyme active site and excessive enzyme jamming along the crowded substrate. Jamming could be alleviated by increasing the decomplexation rate constant but at the expense of reduced processivity. We demonstrate that enhancing the apparent reaction rate required a subtle balance between accelerating the complexation driving force and simultaneously avoiding enzyme jamming. Via a spatiotemporal systems analysis, we developed a unified mechanistic framework that delineates the experimental conditions under which different sets of rate-limiting behaviors emerge. We found that optimization of the complexation-exchange kinetics is critical for overcoming the barriers imposed by interfacial confinement and accelerating the apparent rate of enzymatic cellulose decomposition.
Highlights
Cellobiohydrolase enzymes processively degrade crystalline cellulose into free sugar molecules
We found that optimization of the complexation-exchange kinetics is critical for overcoming the barriers imposed by interfacial confinement and accelerating the apparent rate of enzymatic cellulose decomposition
Increasing the complexation rate constant by 10-fold over the reference rate constant drastically enhanced the conversion of cellulose decomposition in Fig. 3A, but the same increase in the hydrolysis rate constant hardly resulted in any change in the profile
Summary
Cellobiohydrolase enzymes processively degrade crystalline cellulose into free sugar molecules. Results: A spatially resolved kinetic model has been developed to understand the effects of interfacial confinement on cellobiohydrolase activity. Conclusion: Cellobiohydrolase activity is limited by slow rates of complexation with cellulose and traffic jamming among enzymes on the substrate. To understand the impact of spatial constraints on enzyme kinetics, we developed a computational scheme to simulate the reaction network of enzymes with the structures of individual proteins and substrate molecules explicitly resolved in the three-dimensional space. This methodology was applied to elucidate the rate-limiting mechanisms of crystalline cellulose decomposition by cellobiohydrolases. We found that optimization of the complexation-exchange kinetics is critical for overcoming the barriers imposed by interfacial confinement and accelerating the apparent rate of enzymatic cellulose decomposition
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