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

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 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 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 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 de novo protein catalysts in particular, this can provide more enzyme-like catalysts without further rational engineering, computational redesign, or directed evolution

Electric fields are a key determinant of carbapenemase activity in class A β-lactamases

Data repository for "Electric fields are a key determinant of carbapenemase activity in class A β-lactamases" published in ACS Catal. 2024. This repository contains the FieldTools electric field calculation script and the all input files required to reproduce the MD simulations, as well as the MD trajectories generated during this work

Evolution of dynamical networks enhances catalysis in a designer enzyme

Data related to: "Evolution of dynamical networks enhances catalysis in a designer enzyme". H. Adrian Bunzel, J. L. Ross Anderson, Donald Hilvert, Vickery L. Arcus, Marc W. van der Kamp, Adrian J. Mulholland. Nature Chemistry 2021. MD Trajectories of a designed and evolved Kemp eliminase (1A53-2 and 1A53-2.5) in complex with a ground state (GS) or transition state (TS, TS2) model. The ligands are called GS1, TS1, and TS3 in the raw data. Cluster analysis of each trajectory, discriminating between an open (0) or closed (1) state for each frame. Pymol Sessions to reproduce Figures 1-3 of the paper

Emergence of a Negative Activation Heat Capacity during Evolution of a Designed Enzyme

Temperature influences the reaction kinetics and evolvability of all enzymes. To understand how evolution shapes the thermodynamic drivers of catalysis, we optimized the modest activity of a computationally designed enzyme for an elementary proton-transfer reaction by nearly 4 orders of magnitude over 9 rounds of mutagenesis and screening. As theorized for primordial enzymes, the catalytic effects of the original design were almost entirely enthalpic in origin, as were the rate enhancements achieved by laboratory evolution. However, the large reductions in Δ were partially offset by a decrease in Δ and unexpectedly accompanied by a negative activation heat capacity, signaling strong adaptation to the operating temperature. These findings echo reports of temperature-dependent activation parameters for highly evolved natural enzymes and are relevant to explanations of enzymatic catalysis and adaptation to changing thermal environments