Kinetics and thermodynamics of ligand binding to a molten globular enzyme and its native counterpart |
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Authors: | Vamvaca Katherina Jelesarov Ilian Hilvert Donald |
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Institution: | 1 Laboratory of Organic Chemistry, ETH Zurich, Hönggerberg HCI F339, CH-8093 Zurich, Switzerland 2 Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland |
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Abstract: | An engineered monomeric chorismate mutase (mMjCM) has been found to combine high catalytic activity with the characteristics of a molten globule. To gain insight into the dramatic structural changes that accompany binding of a transition-state analog, we examined mMjCM by isothermal calorimetry and compared it with its dimeric parent protein, MjCM (CM from Methanococcus jannaschii), a thermostable and conventionally folded enzyme. As expected for a ligand-induced ordering process, there is a large entropic penalty for binding to the monomer relative to the dimer (− TΔΔS = 5.1 ± 0.5 kcal/mol, at 20 °C). However, this unfavorable entropy term is largely offset by enthalpic gains (ΔΔH = − 3.5 ± 0.4 kcal/mol), presumably arising from tightening of non-covalent interactions throughout the monomeric complex. Stopped-flow kinetic measurements further reveal that the catalytic molten globule binds and releases ligands significantly faster than its natural counterpart, demonstrating that partial structural disorder can speed up molecular recognition. These results illustrate how structural plasticity may strongly perturb the thermodynamics and kinetics of transition-state recognition while negligibly affecting catalytic efficiency. |
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Keywords: | CM chorismate mutase MjCM CM from Methanococcus jannaschii mMjCM engineered CM monomer EcCM CM from Escherichia coli ΔH enthalpic change ΔS entropic change ΔCp heat capacity change ITC isothermal titration calorimetry Kd dissociation constant Ki inhibition constant PBS phosphate-buffered saline |
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