Chemical Mapping Exposes the Importance of Active Site Interactions in Governing the Temperature Dependence of Enzyme Turnover.

Uncovering the role of global protein dynamics in enzyme turnover is needed to fully understand enzyme catalysis. Recently, we have demonstrated that the heat capacity of catalysis, Δ C P ‡ , can reveal links between the protein free energy landscape, global protein dynamics, and enzyme turnover, suggesting that subtle changes in molecular interactions at the active site can affect long-range protein dynamics and link to enzyme temperature activity. Here, we use a model promiscuous enzyme (glucose dehydrogenase from Sulfolobus solfataricus ) to chemically map how individual substrate interactions affect the temperature dependence of enzyme activity and the network of motions throughout the protein. Utilizing a combination of kinetics, red edge excitation shift (REES) spectroscopy, and computational simulation, we explore the complex relationship between enzyme-substrate interactions and the global dynamics of the protein. We find that changes in Δ C P ‡ and protein dynamics can be mapped to specific substrate-enzyme interactions. Our study reveals how subtle changes in substrate binding affect global changes in motion and flexibility extending throughout the protein.