Abstract

The focus of this proposal is to forge a link between protein- dynamics (backbone and side chain) observable by NMR measurements and stimulated by molecular dynamics calculations and the kinetics of enzyme turnover. Two enzymes will serve as paradigms: the dihydrofolate reductase from Escherichia coli and the metallo-beta-lactamase from Bacteroides fragilis. Both enzymes are medically important: dihydrofolate reductase is a key target for chemotherapeutic agents and the metallo-beta-lactamase is a recently evolved enzyme responsible for the resistance of certain strains of pathogenic bacteria to beta-lactam antibiotics. In Project 3, a kinetic scheme will be obtained for turnover of the metallo-beta- lactamase as well as insights into its mechanism of action. Dependent on the findings from NMR studies of dihydrofolate reductase (Project 1) and metallo-beta-lactamase (Project 2) as to the identity of key amino acid residues with unique dynamic motions, site specific mutagenesis of these residues or regions will be performed and the kinetic schemes for both the metallo-beta-lactamase and the reductase redetermined. Departures both kinetic and mechanistic in mature from the behavior of the wild-type enzymes would constitute strong evidence for the importance of protein dynamics in their respective catalytic cycles.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM056879-03
Application #
6301783
Study Section
Project Start
2000-01-01
Project End
2000-12-31
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
3
Fiscal Year
2000
Total Cost
$147,723
Indirect Cost

  Institution

Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037

  Publications

Boehr, David D; Dyson, H Jane; Wright, Peter E (2006) An NMR perspective on enzyme dynamics. Chem Rev 106:3055-79
Boehr, David D; McElheny, Dan; Dyson, H Jane et al. (2006) The dynamic energy landscape of dihydrofolate reductase catalysis. Science 313:1638-42
Thorpe, Ian F; Brooks 3rd, Charles L (2005) Conformational substates modulate hydride transfer in dihydrofolate reductase. J Am Chem Soc 127:12997-3006
McElheny, Dan; Schnell, Jason R; Lansing, Jonathan C et al. (2005) Defining the role of active-site loop fluctuations in dihydrofolate reductase catalysis. Proc Natl Acad Sci U S A 102:5032-7
Venkitakrishnan, Rani P; Zaborowski, Eduardo; McElheny, Dan et al. (2004) Conformational changes in the active site loops of dihydrofolate reductase during the catalytic cycle. Biochemistry 43:16046-55
Chen, Jianhan; Brooks 3rd, Charles L; Wright, Peter E (2004) Model-free analysis of protein dynamics: assessment of accuracy and model selection protocols based on molecular dynamics simulation. J Biomol NMR 29:243-57
Schnell, Jason R; Dyson, H Jane; Wright, Peter E (2004) Structure, dynamics, and catalytic function of dihydrofolate reductase. Annu Rev Biophys Biomol Struct 33:119-40
Schnell, Jason R; Dyson, H Jane; Wright, Peter E (2004) Effect of cofactor binding and loop conformation on side chain methyl dynamics in dihydrofolate reductase. Biochemistry 43:374-83
Thorpe, Ian F; Brooks 3rd, Charles L (2004) The coupling of structural fluctuations to hydride transfer in dihydrofolate reductase. Proteins 57:444-57
Osborne, Michael J; Venkitakrishnan, Rani P; Dyson, H Jane et al. (2003) Diagnostic chemical shift markers for loop conformation and substrate and cofactor binding in dihydrofolate reductase complexes. Protein Sci 12:2230-8

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