Observed live: Water is an active team player for enzymes
RUB researchers report in Nature Structural & Molecular Biology
Water acts as an "adhesive" in biological enzyme substrate compounds
Which role does the solvent play?
Enzymes are natural substances accelerating and controlling the metabolic processes in the body. They are, for example, of central importance for the immune system, as they control the balance between activating and inhibiting immune reactions and play an important role in inflammation processes. It had been known for some time that enzymatic functions take place in various solvents at highly differing speeds. But up to now, the contribution made by the solvent - this is water in biological processes - on a molecular level had not yet been clarified.
Two new techniques combined
Prof. Havenith’s group at the RUB and Prof. Irit Sagi’s group at the Weizmann Institute have combined two newly developed experimental techniques, in order to directly study the role of water in enzymatic reactions utilizing different peptide substrates designed by the group of Prof. Gregg Fields. The enzyme they studied was Matrix Metalloproteinase (MMPs) which belongs to an important class of enzymes known to promote cancer metastasis, inflammatory and autoimmune diseases by remodeling tissues or extracellular matrices. Thus, the broad spectrum of applications makes this family of enzymes an important target for rational drug design. "The role of water for the molecular mechanisms driving catalysis of MMPs or any other enzyme is not known" says Prof. Havenith.
Precise characterisation of all "players"
In order to obtain a detailed understanding of the reaction the researchers looked at all constituents which are involved: The MMP enzyme, the protein and the water as as solvent. In the experiment, the scientists investigated the binding of the peptide substrate to the MMP. With the help of time-resolved X-ray spectroscopy they were able to characterize precisely the structural changes in the vicinity of the active site of the enzyme (here: of the zinc atom) with atomic resolution. With the help of kinetic THz absorption spectroscopy (KITA) they recorded the changes of the fast water motions in real-time.
The role of water for future drug design
In representative MMP-substrate combinations, correlation was found between the fluctuations of the water network the structural changes and the biological function. Molecular dynamic simulations provided an explanation for the observations: While the substrate has not found yet its specific binding site of the enzyme, the water dynamism, i.e. the opening and reformation of hydrogen bonds between water molecules (the "terahertz dance" of the water), is fast. At the same time as the substrate is docking to the active site, the water motions in the vicinity of the protein are slowed down. This change of the THz dance of the water with the formation of the enzyme-substrate binding is however exclusively observed in biologically active enzyme-substrate combinations. "The retardation of the water dynamism, observed for the first time, thus seems to be an essential part of the functional control", says Prof. Havenith. "Therefore, in future, taking the role of the water into account in the development of therapeutic agents targeting these enzymes in vivo might become of importance.“
This work is part of "Solvation Science@RUB“, the research topic of the new center of molecular spectroscopy and simulation of solvent controlled processes at the RUB (ZEMOS), and of the excellence cluster application of the RUB “RESOLV”, which is now under review at the German council of science. In chemistry, process engineering and biology, there are an enormous number of publications describing solvents as inert (passive) media for molecular processes. Beyond this traditional view, the active role of the solvent is however becoming more and more visible. New experimental and theoretical methods now permit investigation, description and systematic control of the structure, dynamism and kinetics of complex solvation phenomena on a molecular level. "So it is now most timely to develop general models with a predictive power for solvation processes", says Prof. Havenith. Precisely that is the objective of "Solvation Science@RUB".
This work has been supported by the Robert A. Welch Foundation, US National Institutes of Health grant, Israel Science Foundation, and the VW Stiftung and by the Ruhr University Research School.
M. Grossman, B. Born, M. Heyden, D. Tworowski, G. Fields, I. Sagi, M. Havenith: Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site. Nature Structural & Molecular Biology, Advance Online Publication (AOP), doi: 10.1038/nsmb.2120