Visualizing Structures, Dynamics and Function of Enzymes

Abstract

In order to understand how enzymes work it is required to know about its conformational landscape, its enzymatic state or interaction with the substrate or product, and the exchange times between conformations and enzymatic states. Experimentally this is extremely challenging because it requires to sample over large time domains, as well as resolving low transiently populated states. Using a hybrid approach involving multiparameter fluorescence spectroscopy, multiscale computer simulations and mutagenesis we are able to map the conformational landscape involving three conformational states (C1, C2, and C3) that are sampled from ns to ms. Furthermore, we present a state matrix that links the enzyme state of the bacteriophage T4 (T4L) as it follows a Michaelis-Menten like formalism. In all we identify T4L as a three state system linking each of the fundamental steps in enzyme function: Substrate binding in C1, catalysis in C2, and product release in C3. We validate the conformational state C1 against the crystallographic structure with identification code in the protein data bank (PDBID) 172L with an accuracy of 1.8 A RMSD and 2σ precision of 0.6 A. The C2 agrees very well with the PDBID 148L with an accuracy of 2.1 A RMSD and 2σ precision of 0.9 A. The conformational state C3 has not been identified before and has a 2σ precision of 2.6 A. Our findings for T4L demonstrate that a fine-tuned shift of conformational equilibrium leads to motions of active cleaning where the energies of substrate binding and product formation drive the conformational equilibrium. The directionality of the overall process is manifested by the free reaction enthalpy of the glycosidic bond cleavage. In all, our work highlights the potential of our hybrid approach to observe thermally accessible, low-populated intermediates with high spatial and temporal resolution.