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Rigidifying a De Novo Enzyme Increases Activity and Induces a Negative Activation Heat Capacity.

Hindson, Sarah A and Bunzel, H Adrian and Frank, Bettina and Svistunenko, Dimitri A and Williams, Christopher and van der Kamp, Marc W and Mulholland, Adrian J and Pudney, Christopher R and Anderson, JL Ross (2021) 'Rigidifying a De Novo Enzyme Increases Activity and Induces a Negative Activation Heat Capacity.' ACS Catalysis, 11 (18). pp. 11532-11541. ISSN 2155-5435

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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 <i>de novo</i> peroxidase C45. We demonstrate that 2,2,2-triflouoroethanol (TFE) affects the equilibrium of enzyme conformational states, tending toward a more globally rigid structure. This is correlated with increases in 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 states. We compare the thermodynamics of the <i>de novo</i> enzyme activity to those of a natural peroxidase, horseradish peroxidase. We find that the native enzyme resembles the rigidified <i>de novo</i> enzyme in terms of the thermodynamics of enzyme catalysis and the putative distribution of protein dynamics between the ground and transition states. 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 <i>de novo</i> protein catalysts in particular, this can provide more enzyme-like catalysts without further rational engineering, computational redesign, or directed evolution.

Item Type: Article
Uncontrolled Keywords: enzyme catalysis; protein dynamics; peroxidase; C45; MMRT; REES; activation heat capacity
Divisions: Faculty of Science and Health
Faculty of Science and Health > Life Sciences, School of
SWORD Depositor: Elements
Depositing User: Elements
Date Deposited: 14 Dec 2021 11:42
Last Modified: 18 Aug 2022 10:41
URI: http://repository.essex.ac.uk/id/eprint/31888

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