Defining the Role of Active-Site Loop Fluctuations in Dihydrofolate Reductase Catalysis

Dynamic processes are implicit in the catalytic function of all enzymes. To obtain insights into the relationship between the dynamics and thermodynamics of protein fluctuations and catalysis, we have measured millisecond time scale motions in the enzyme dihydrofolate reductase using NMR relaxation...

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Bibliographic Details
Published in:	Proceedings of the National Academy of Sciences - PNAS Vol. 102; no. 14; pp. 5032 - 5037
Main Authors:	McElheny, Dan, Schnell, Jason R., Lansing, Jonathan C., Dyson, H. Jane, Wright, Peter E., Petsko, Gregory A.
Format:	Journal Article
Language:	English
Published:	United States National Academy of Sciences 05-04-2005 National Acad Sciences
Subjects:	Active sites Binding sites Biochemistry Biological Sciences Biophysical Phenomena Biophysics Catalysis Catalytic Domain Chemical equilibrium Conformity Enzymes Escherichia coli - enzymology Folic Acid - metabolism Ground state Hydrides Kinetics Models, Biological Models, Molecular NADP - metabolism NMR Nuclear magnetic resonance Nuclear Magnetic Resonance, Biomolecular Protein Conformation Tetrahydrofolate Dehydrogenase - chemistry Tetrahydrofolate Dehydrogenase - metabolism Thermodynamics
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Summary:	Dynamic processes are implicit in the catalytic function of all enzymes. To obtain insights into the relationship between the dynamics and thermodynamics of protein fluctuations and catalysis, we have measured millisecond time scale motions in the enzyme dihydrofolate reductase using NMR relaxation methods. Studies of a ternary complex formed from the substrate analog folate and oxidized NADP+cofactor revealed conformational exchange between a ground state, in which the active site loops adopt a closed conformation, and a weakly populated (4.2% at 30°C) excited state with the loops in the occluded conformation. Fluctuations between these states, which involve motions of the nicotinamide ring of the cofactor into and out of the active site, occur on a time scale that is directly relevant to the structural transitions involved in progression through the catalytic cycle.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Abbreviations: CPMG, Carr–Purcell–Meiboom–Gill; DHFR, dihydrofolate reductase; THF, tetrahydrofolate. Present address: Division of Biological Sciences, University of Chicago, Chicago, IL 60637. Author contributions: P.E.W. designed research; D.M. and J.R.S. performed research; J.C.L. contributed new reagents/analytic tools; D.M., J.R.S., H.J.D., and P.E.W. analyzed data; and D.M., H.J.D., and P.E.W. wrote the paper. This paper was submitted directly (Track II) to the PNAS office. To whom correspondence should be addressed. E-mail: wright@scripps.edu. Present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115. Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved March 2, 2005
ISSN:	0027-8424 1091-6490
DOI:	10.1073/pnas.0500699102