Linkage between catalytic mechanism and conformational dynamics in (beta/alpha)8-barrel enzymes

• Applicant: Professor Dr. Reinhard Sterner
• Subject Area: Biochemistry ; Biophysics ; Structural Biology
• Term: from 2015 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 273747520

The catalytic power of enzymes is linked with its conformational dynamics, but the molecular determinants of this linkage are largely unclear. Here, we address this problem by analyzing in parallel the kinetic mechanisms and the dynamics of loops near the active sites of enzymes. We will use the (beta/alpha)8-barrel proteins indoleglycerol phosphate synthase (IGPS) and the cyclase subunit (HisF) of imidazole glycerol phosphate synthase (ImGPS) from mesophilic and thermophilic microorganisms. For the thermophilic and the mesophilic enzymes, the rates of the individual catalytic steps, substrate and product binding and release, chemical transformation, will be determined by using pre-steady state kinetic measurements at various temperatures to obtain a detailed picture of the enzymatic mechanisms. The information deduced from these experiments will then be correlated with the dynamics of loop movements. To this end, the beta1alpha1-loop at the active site of mesophilic and thermophilic IGPS will be spin labeled, and changes in its dynamics in the course of catalysis will be followed by EPR. For mesophilic and thermophilic HisF, motions of the beta/alpha-loops near the active site will be analyzed by NMR relaxation dispersion experiments. Our results should allow us to gain novel insight into the molecular nature of the coupling between protein dynamics and the crossing of energy barriers in enzyme catalysis. They should also reveal whether and how homologous mesophilic and thermophilic (beta/alpha)8-barrel enzymes differ in their catalytic strategies. A better understanding of the relationship between dynamics and catalytic efficiency will also be valuable for enzyme engineering.

• DFG Programme: Research Grants
• Co-Investigators: Professor Dr. Remco Sprangers; Professor Dr. Heinz-Jürgen Steinhoff