
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.08.23.609221
preprint
1
Article
A structural perspective on the temperature-dependent activity of enzymes
McLeod Matthew J. http://orcid.org/0000-0001-5540-916X

Barwell Sarah A. E. http://orcid.org/0000-0002-5703-5525

Holyoak Todd http://orcid.org/0000-0002-7329-1115

Thorne Robert Edward
23 8 2024
2024.08.23.609221https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.08.23.609221
nihpp-2024.08.23.609221.pdf
ABSTRACT

Enzymes are biomolecular catalysts whose activity varies with temperature. Unlike for small-molecule catalysts, the structural ensembles of enzymes can vary substantially with temperature, and it is in general unclear how this modulates the temperature dependence of activity. Here multi-temperature X-ray crystallography was used to record structural changes from −20°C to 40°C for a mesophilic enzyme in complex with inhibitors mimicking substrate-, intermediate-, and product-bound states, representative of major complexes underlying the kinetic constant k cat . Both inhibitors, substrates and catalytically relevant loop motifs increasingly populate catalytically competent conformations as temperature increases. These changes occur even in temperature ranges where kinetic measurements show roughly linear Arrhenius/Eyring behavior where parameters characterizing the system are assumed to be temperature independent. Simple analysis shows that linear Arrhenius/Eyring behavior can still be observed when the underlying activation energy / enthalpy values vary with temperature, e.g., due to structural changes, and that the underlying thermodynamic parameters can be far from values derived from Arrhenius/Eyring model fits. Our results indicate a critical role for temperature-dependent atomic-resolution structural data in interpreting temperature-dependent kinetic data from enzymatic systems.

One-Sentence Summary

Structural data spanning a 60°C temperature range for enzyme complexes mimicking the substrate-, intermediate-, and product-bound states illuminate how small temperature-dependent structural changes may modulate activity and render parameters deduced from Arrhenius/Eyring plots unreliable.
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