Conformational relaxation following hydride transfer plays a limiting role in dihydrofolate reductase catalysis

David D. Boehr, H. Jane Dyson, Peter E. Wright

Research output: Contribution to journalArticlepeer-review

49 Scopus citations

Abstract

The catalytic cycle of an enzyme is frequently associated with conformational changes that may limit maximum catalytic throughput. In Escherichia coli dihydrofolate reductase, release of the tetrahydrofolate (THF) product is the rate-determining step under physiological conditions and is associated with an "occluded" to "closed" conformational change. In this study, we demonstrate that in dihydrofolate reductase the closed to occluded conformational change in the product ternary complex (E·THF·NADP+) also gates progression through the catalytic cycle. Using NMR relaxation dispersion, we have measured the temperature and pH dependence of microsecond to millisecond time scale backbone dynamics of the occluded E·THF·NADP+ complex. Our studies indicate the presence of three independent dynamic regions, associated with the active-site loops, the cofactor binding cleft, and the C-terminus and an adjacent loop, which fluctuate into discrete conformational substates with different kinetic and thermodynamic parameters. The dynamics of the C-terminally associated region is pH-dependent (pKa < 6), but the dynamics of the active-site loops and cofactor binding cleft are pH-independent. The active-site loop dynamics access a closed conformation, and the accompanying closed to occluded rate constant is comparable to the maximum pH-independent hydride transfer rate constant. Together, these results strongly suggest that the closed to occluded conformational transition in the product ternary complex is a prerequisite for progression through the catalytic cycle and that the rate of this process places an effective limit on the maximum rate of the hydride transfer step.

Original languageEnglish (US)
Pages (from-to)9227-9233
Number of pages7
JournalBiochemistry
Volume47
Issue number35
DOIs
StatePublished - Sep 2 2008

All Science Journal Classification (ASJC) codes

  • Biochemistry

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