LIFE SCIENCES

Protein-substrate interactions are crucial for enzymatic activity. In the substrate binding site of an enzyme, the substrate is expected to bind in a productive orientation in close proximity to the active site residues of the enzyme. Scientists of the Institute of Biochemistry and Technical Biochemistry of the University of Stuttgart developed a simulation method to model substrate access to the active site of esterases, lipases, and acyl transferases. There is increasing experimental evidence that substrate access to and product exit from the binding site might become rate limiting steps under realistic reaction conditions. To study the concentration dependency of substrate access at high substrate concentrations. A series of simulations were performed for the enzymes with different substrates at varying concentrations, and the free energy profile was determined for substrate binding as a function of the distance of a substrate molecule from the active site of the enzyme.

A typical substrate of Candida antarctica lipase B is 4-nitrophenyl butyrate (Fig. 1). The free energy profile demonstrated that at distances beyond 7 nm, the substrate does not interact with the enzyme, but binds to the protein surface and the substrate binding pocket, indicated by a drop of the free energy by 3.5 kT below a distance of 3.3 nm. Free energy profiles are a valuable tool for a better understanding of substrate access, to identify hotspots for protein engineering, and to support process design.