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Protein function often involves changes between different conformations.Central questions are how these conformational changes are coupled to thebinding or catalytic processes during which they occur, and how they affect thecatalytic rates of enzymes. An important model system is the enzymedihydrofolate reductase (DHFR) from E. coli, which exhibits characteristicconformational changes of the active-site loop during the catalytic step andduring unbinding of the product. In this article, we present a general kineticframework that can be used (1) to identify the ordering of events in thecoupling of conformational changes, binding and catalysis and (2) to determinethe rates of the substeps of coupled processes from a combined analysis of NMRR2 relaxation dispersion experiments and traditional enzyme kineticsmeasurements. We apply this framework to E. coli DHFR and find that theconformational change during product unbinding follows aconformational-selection mechanism, i.e. the conformational change occurspredominantly prior to unbinding. The conformational change during thecatalytic step, in contrast, is an induced change, i.e. the change occurs afterthe chemical reaction. We propose that the reason for these conformationalchanges, which are absent in human and other vertebrate DHFRs, is robustness ofthe catalytic rate against large pH variations and changes to substrate/productconcentrations in E. coli.

Conformational selection and induced changes along the catalytic cycle of E. coli DHFR