Conformational selection and induced changes along the catalytic cycle of Escherichia coli dihydrofolate reductase.
Protein function often involves changes between different conformations. Central questions are how these conformational changes are coupled to the binding or catalytic processes during which they occur, and how they affect the catalytic rates of enzymes. An important model system is the enzyme dihydrofolate reductase (DHFR) from Escherichia coli, which exhibits characteristic conformational changes of the active-site loop during the catalytic step and during unbinding of the product. In this article, we present a general kinetic framework that can be used (1) to identify the ordering of events in the coupling of conformational changes, binding, and catalysis and (2) to determine the rates of the substeps of coupled processes from a combined analysis of nuclear magnetic resonance R(2) relaxation dispersion experiments and traditional enzyme kinetics measurements. We apply this framework to E. coli DHFR and find that the conformational change during product unbinding follows a conformational-selection mechanism, that is, the conformational change occurs predominantly prior to unbinding. The conformational change during the catalytic step, in contrast, is an induced change, that is, the change occurs after the chemical reaction. We propose that the reason for these conformational changes, which are absent in human and other vertebrate DHFRs, is robustness of the catalytic rate against large pH variations and changes to substrate/product concentrations in E. coli.
Max Planck Institute of Colloids and Interfaces, Department of Theory and Bio-Systems, Science Park Golm, 14424 Potsdam, Germany. weikl@mpikg.mpg.de
Thomas R
TR
Weikl
David D
DD
Boehr
D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013763:Tetrahydrofolates; D013816:Thermodynamics
D029968:Escherichia coli Proteins; D013763:Tetrahydrofolates; C030371:5,6,7,8-tetrahydrofolic acid; D013762:Tetrahydrofolate Dehydrogenase