Rigidifying a de novo enzyme increases activity and induces a negative activation heat capacity

Sarah Hindson, Hans Bunzel, Bettina Frank, Dimitri Svistunenko, Christopher Williams, Marc W van der Kamp, Adrian Mulholland, Christopher Pudney, Ross Anderson

Research output: Contribution to journalArticlepeer-review

Abstract

Conformational sampling profoundly impacts the overall activity and temperature dependence of enzymes. Peroxidases have emerged as versatile platforms for high value biocatalysis owing to their broad palette of potential biotransformations. Here, we explore the role of conformational sampling in mediating activity in the de novo peroxidase C45. We demonstrate that 2,2,2-triflouoroethanol (TFE) affects the equilibrium of enzyme conformational states, tending towards a more globally rigid structure. This is correlated with increases both stability and activity. Notably, these effects are concomitant with the emergence of curvature in the temperature-activity profile, trading off activity gains at ambient temperature with losses at high temperatures. We apply macromolecular rate theory (MMRT) to understand enzyme temperature dependence data. These data point to an increase in protein rigidity associated with a difference in the distribution of protein dynamics between the ground and transition state. We compare the thermodynamics of the de novo enzyme activity to those of a natural peroxidase, horseradish peroxidase. We find that the native enzyme resembles the rigidified de novo enzyme in terms of the thermodynamics of enzyme catalysis and the putative distribution of protein dynamics between the ground and transition state. The addition of TFE apparently causes C45 to behave more like the natural enzyme. Our data suggest robust, generic strategies for improving biocatalytic activity by manipulating protein rigidity; for functional de novo protein catalysts in particular, this can provide more enzyme-like catalysts without further rational engineering, computational redesign or directed evolution.
Original languageEnglish
Pages (from-to)11532-11541
Number of pages10
JournalACS Catalysis
Volume11
Issue number18
Early online date1 Sep 2021
DOIs
Publication statusPublished - 17 Sep 2021

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