Cooperative Conformational Transitions Underpin the Activation Heat Capacity in the Temperature Dependence of Enzyme Catalysis

Emma J Walker; Carlin J Hamill; Rory Crean; Michael S Connolly; Annmaree K Warrender; Kirsty L Kraakman; Erica J Prentice; Alistair Steyn-Ross; Moira Steyn-Ross; Christopher R Pudney; Marc W van der Kamp; Louis A Schipper; Adrian J Mulholland; Vickery L Arcus

doi:10.1021/acscatal.3c05584

Cooperative Conformational Transitions Underpin the Activation Heat Capacity in the Temperature Dependence of Enzyme Catalysis

ACS Catal. 2024 Mar 8;14(7):4379-4394. doi: 10.1021/acscatal.3c05584. eCollection 2024 Apr 5.

Affiliations

¹ Te Aka Ma̅tuatua School of Science, University of Waikato, Hamilton 3214, New Zealand.
² Centre for Computational Chemistry, School of Chemistry, University of Bristol, Bristol BS8 1TS, U.K.
³ School of Engineering, University of Waikato, Hamilton 3214, New Zealand.
⁴ Department of Biology and Biochemistry, Centre for Biosensors, Bioelectronics and Biodevices, University of Bath, Bath ST16 2TB, U.K.
⁵ School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, U.K.

Abstract

Many enzymes display non-Arrhenius behavior with curved Arrhenius plots in the absence of denaturation. There has been significant debate about the origin of this behavior and recently the role of the activation heat capacity (ΔC_P^⧧) has been widely discussed. If enzyme-catalyzed reactions occur with appreciable negative values of ΔC_P^⧧ (arising from narrowing of the conformational space along the reaction coordinate), then curved Arrhenius plots are a consequence. To investigate these phenomena in detail, we have collected high precision temperature-rate data over a wide temperature interval for a model glycosidase enzyme MalL, and a series of mutants that change the temperature-dependence of the enzyme-catalyzed rate. We use these data to test a range of models including macromolecular rate theory (MMRT) and an equilibrium model. In addition, we have performed extensive molecular dynamics (MD) simulations to characterize the conformational landscape traversed by MalL in the enzyme-substrate complex and an enzyme-transition state complex. We have crystallized the enzyme in a transition state-like conformation in the absence of a ligand and determined an X-ray crystal structure at very high resolution (1.10 Å). We show (using simulation) that this enzyme-transition state conformation has a more restricted conformational landscape than the wildtype enzyme. We coin the term "transition state-like conformation (TLC)" to apply to this state of the enzyme. Together, these results imply a cooperative conformational transition between an enzyme-substrate conformation (ES) and a transition-state-like conformation (TLC) that precedes the chemical step. We present a two-state model as an extension of MMRT (MMRT-2S) that describes the data along with a convenient approximation with linear temperature dependence of the activation heat capacity (MMRT-1L) that can be used where fewer data points are available. Our model rationalizes disparate behavior seen for MalL and previous results for a thermophilic alcohol dehydrogenase and is consistent with a raft of data for other enzymes. Our model can be used to characterize the conformational changes required for enzyme catalysis and provides insights into the role of cooperative conformational changes in transition state stabilization that are accompanied by changes in heat capacity for the system along the reaction coordinate. TLCs are likely to be of wide importance in understanding the temperature dependence of enzyme activity and other aspects of enzyme catalysis.