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How enzymes achieve their enormous rate enhancements remains a central question in biology, and our understanding to date has impacted drug development, influenced enzyme design, and deepened our appreciation of evolutionary processes. While enzymes position catalytic and reactant groups in active sites, physics requires that atoms undergo constant motion. Numerous proposals have invoked positioning or motions as central for enzyme function, but a scarcity of experimental data has limited our understanding of positioning and motion, their relative importance, and their changes through the enzyme’s reaction cycle. To examine positioning and motions and test catalytic proposals, we collected “room temperature” X-ray crystallography data forPseudomonas putidaketosteroid isomerase (KSI), and we obtained conformational ensembles for this and a homologous KSI from multiple PDB crystal structures. Ensemble analyses indicated limited change through KSI’s reaction cycle. Active site positioning was on the 1- to 1.5-Å scale, and was not exceptional compared to noncatalytic groups. The KSI ensembles provided evidence against catalytic proposals invoking oxyanion hole geometric discrimination between the ground state and transition state or highly precise general base positioning. Instead, increasing or decreasing positioning of KSI’s general base reduced catalysis, suggesting optimized Ångstrom-scale conformational heterogeneity that allows KSI to efficiently catalyze multiple reaction steps. Ensemble analyses of surrounding groups for WT and mutant KSIs provided insights into the forces and interactions that allow and limit active-site motions. Most generally, this ensemble perspective extends traditional structure–function relationships, providing the basis for a new era of “ensemble–function” interrogation of enzymes.
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Ensemble–function relationships to evaluate catalysis in the ketosteroid isomerase oxyanion hole
Following decades of insights from structure–function studies, there is now a need to progress from a static to dynamic view of enzymes. Comparison of prior cryo X-ray structures suggested that deleterious effects from ketosteroid isomerase (KSI) mutants arise from misalignment of the oxyanion hole catalytic residue, Y16. However, multi-conformer models from room temperature X-ray diffraction revealed an ensemble of Y16 conformers indistinguishable from WT for Y32F/Y57F KSI and a distinct, non-native ensemble for Y16 in Y57F KSI. Functional analyses suggested rate effects arise from weakened hydrogen bonding, due to disruption of the Y16/Y57/Y32 hydrogen bond network, and repositioning of the general base. In general, catalytic changes can be deconvoluted into effects on the probability of occupying a state (P-effects) and the reactivity of each state (k-effects). Our results underscore the need for ensemble–function analysis to decipher enzyme function and ultimately manipulate their extraordinary capabilities.
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- Award ID(s):
- 1714723
- PAR ID:
- 10355636
- Date Published:
- Journal Name:
- Science advances
- ISSN:
- 2375-2548
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1101/2021.09.29.461692
