A Dynamic Knockout Reveals That Conformational Fluctuations Influence the Chemical Step of Enzyme Catalysis
Abstract
Conformational dynamics play a key role in enzyme catalysis. Although protein motions have clear implications for ligand flux, a role for dynamics in the chemical step of enzyme catalysis has not been clearly established. We generated a mutant of Escherichia coli dihydrofolate reductase that abrogates millisecond-time-scale fluctuations in the enzyme active site without perturbing its structural and electrostatic preorganization. This dynamic knockout severely impairs hydride transfer. Thus, we have found a link between conformational fluctuations on the millisecond time scale and the chemical step of an enzymatic reaction, with broad implications for our understanding of enzyme mechanisms and for design of novel protein catalysts.
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References and Notes
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Materials and methods are available as supporting material on Science Online.
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Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. In the mutants, other amino acids were substituted at certain locations; for example, H134R indicates that histidine at position 134 was replaced by arginine.
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The terminal methyl of M20 is not clearly defined by the electron density in 1RX2 and in 1RX2–re-refined, and is probably flexible. In addition, density for water 47 is observed in 1RX2–re-refined but was not built into 1RX2. A discussion on building M20 and water47 into 1RX2-rerefined is included in the supporting online material (SOM) text.
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Information
Published In
Science
Volume 332 | Issue 6026
8 April 2011
8 April 2011
Copyright
Copyright © 2011, American Association for the Advancement of Science.
Submission history
Received: 1 October 2010
Accepted: 16 February 2011
Published in print: 8 April 2011
Acknowledgments
The authors acknowledge L. Tuttle for invaluable discussions, S. Bae for NMR data on the S148A mutant, H. Tien and D. Marciano of the Robotics Core at the Joint Center for Structural Genomics (JCSG) for automated crystal screening, and X. Dai for technical assistance with crystallographic data collection. This work was supported by the National Institutes of Health (NIH) grant GM75995 and the Skaggs Institute of Chemical Biology. The JCSG is supported by NIH National Institute of General Medical Sciences (NIGMS) (U54 GM094586). D.C.E. is supported by a predoctoral fellowship from the Achievement Rewards for College Scientists Foundation and grant GM080209 from the NIH Molecular Evolution Training Program. The GM/CA CAT 23-ID-D has been funded in whole or in part with federal funds from National Cancer Institute (Y1-CO-1020) and NIGMS (Y1-GM-1104). Use of the Advanced Photon Source (APS) was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, under contract DE-AC02-06CH11357. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIGMS or NIH. Coordinates and structure factors of N23PP/S148A ecDHFR and 1RX2–re-refined are deposited in PDB (code 3QL0 and 3QL3). G.B. and P.E.W. designed the research. G.B. performed NMR experiments. G.B. and D.C.E. performed crystallography experiments and structure refinement. I.A.W. supervised structure refinement. J.L. and J.G. performed kinetic experiments and analysis. G.B., P.E.W., and S.J.B. analyzed and interpreted the data. G.B., P.E.W., and J.L. wrote the manuscript. I.A.W., H.J.D., and S.J.B. edited the manuscript.
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