Short talk:
Relation between loop motion and catalytic activity by the example of the (βα)8-barrel enzyme HisF

Sandra Schlee1, Hupfeld Enrico1, Wurm Jan-Philip1, Sprangers Remco1, Sterner Reinhard1

1Institute of Biophysics and Physical Biochemistry, Regensburg Center for Biochemistry, University of Regensburg, Regensburg, Germany

Motions of active site loops are essential for catalytic activity. Enzymes with the prevalent (βα)8- barrel fold are highly suitable for studying the relationship between loop dynamics and catalysis. We analysed the β1α1-loop of the cyclase subunit HisF of imidazole glycerol phosphate synthase which catalyses the formation of the imidazole ring in histidine biosynthesis. During the catalytic cycle this loop undergoes a transformation from a flexible, entropically rich form into a stiff ligand-bound conformation. The aim of this work was to define the functional significance of β1α1-loop mobility and its structural transitions for the catalytic activity of HisF. Steady-state kinetics showed that certain amino acid substitutions within the β1α1-loop reduce the turnover velocity of the enzymatic reaction, whereas substrate affinity is hardly affected. In- depth analyses of HisF mutants with altered loop dynamics did not evince a direct correlation between loop flexibility and turnover velocity. Instead, NMR spectroscopy revealed that catalytically active HisF mutants bind substrate via an induced-fit mechanism involving the β1α1-loop. Stopped-flow analysis under single- and multiple-turnover conditions suggested that turnover velocity of HisF is not limited by product release. In addition, substrate binding and product release steps are fast in comparison to the turnover number kcat, rendering the chemical conversion reaction as the rate-determining step in the catalytic cycle. Hence, although the structural changes of the β1α1-loop associated with the transition from the inactive to active conformation are not rate-limiting for catalysis, the induced-fit motion is a prerequisite for effective enzymatic function.

Go back