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bioRxiv · 10.1101/2021.09.29.461692

Ensemble-function relationships to evaluate catalysis in the ketosteroid isomerase oxyanion hole

Yabukarski, F.·Doukov, T.·Pinney, M.·Biel, J.·Fraser, J.·Herschlag, D.
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Abstract

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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Yabukarski, F., Doukov, T., Pinney, M., Biel, J., Fraser, J., Herschlag, D.. 2021-09-30. Ensemble-function relationships to evaluate catalysis in the ketosteroid isomerase oxyanion hole. https://doi.org/10.1101/2021.09.29.461692

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