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Title: Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase

Abstract

Homotetrameric R67 dihydrofolate reductase possesses 222 symmetry and a single active site pore. This situation results in a promiscuous binding site that accommodates either the substrate, dihydrofolate (DHF), or the cofactor, NADPH. NADPH interacts more directly with the protein as it is larger than the substrate. In contrast, the p-aminobenzoyl-glutamate tail of DHF, as monitored by nuclear magnetic resonance and crystallography, is disordered when bound. To explore whether smaller active site volumes (which should decrease the level of tail disorder by confinement effects) alter steady state rates, asymmetric mutations that decreased the half-pore volume by ~35% were constructed. Only minor effects on k cat were observed. To continue exploring the role of tail disorder in catalysis, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-mediated cross-linking between R67 DHFR and folate was performed. A two-folate, one-tetramer complex results in the loss of enzyme activity where two symmetry-related K32 residues in the protein are cross-linked to the carboxylates of two bound folates. The tethered folate could be reduced, although with a ≤ 30-fold decreased rate, suggesting decreased dynamics and/or suboptimal positioning of the cross-linked folate for catalysis. Computer simulations that restrain the dihydrofolate tail near K32 indicate that cross-linking still allows movement of the p-aminobenzoyl ring, which allows themore » reaction to occur. Finally, a bis-ethylene-diamine-α,γ-amide folate adduct was synthesized; both negatively charged carboxylates in the glutamate tail were replaced with positively charged amines. The K i for this adduct was ~9-fold higher than for folate. These various results indicate a balance between folate tail disorder, which helps the enzyme bind substrate while dynamics facilitates catalysis.« less

Authors:
 [1];  [1];  [2];  [3];  [1]
  1. Department of Biochemistry &, Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0840, United States
  2. Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, United States
  3. Department of Biochemistry &, Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0840, United States; Computer Science and Mathematics Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Oak Ridge Leadership Computing Facility (OLCF)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1565444
Resource Type:
Journal Article
Journal Name:
Biochemistry
Additional Journal Information:
Journal Volume: 55; Journal Issue: 1; Journal ID: ISSN 0006-2960
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
Biochemistry & Molecular Biology

Citation Formats

Duff, Michael R., Chopra, Shaileja, Strader, Michael Brad, Agarwal, Pratul K., and Howell, Elizabeth E. Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase. United States: N. p., 2015. Web. doi:10.1021/acs.biochem.5b00981.
Duff, Michael R., Chopra, Shaileja, Strader, Michael Brad, Agarwal, Pratul K., & Howell, Elizabeth E. Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase. United States. doi:10.1021/acs.biochem.5b00981.
Duff, Michael R., Chopra, Shaileja, Strader, Michael Brad, Agarwal, Pratul K., and Howell, Elizabeth E. Mon . "Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase". United States. doi:10.1021/acs.biochem.5b00981.
@article{osti_1565444,
title = {Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase},
author = {Duff, Michael R. and Chopra, Shaileja and Strader, Michael Brad and Agarwal, Pratul K. and Howell, Elizabeth E.},
abstractNote = {Homotetrameric R67 dihydrofolate reductase possesses 222 symmetry and a single active site pore. This situation results in a promiscuous binding site that accommodates either the substrate, dihydrofolate (DHF), or the cofactor, NADPH. NADPH interacts more directly with the protein as it is larger than the substrate. In contrast, the p-aminobenzoyl-glutamate tail of DHF, as monitored by nuclear magnetic resonance and crystallography, is disordered when bound. To explore whether smaller active site volumes (which should decrease the level of tail disorder by confinement effects) alter steady state rates, asymmetric mutations that decreased the half-pore volume by ~35% were constructed. Only minor effects on kcat were observed. To continue exploring the role of tail disorder in catalysis, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-mediated cross-linking between R67 DHFR and folate was performed. A two-folate, one-tetramer complex results in the loss of enzyme activity where two symmetry-related K32 residues in the protein are cross-linked to the carboxylates of two bound folates. The tethered folate could be reduced, although with a ≤ 30-fold decreased rate, suggesting decreased dynamics and/or suboptimal positioning of the cross-linked folate for catalysis. Computer simulations that restrain the dihydrofolate tail near K32 indicate that cross-linking still allows movement of the p-aminobenzoyl ring, which allows the reaction to occur. Finally, a bis-ethylene-diamine-α,γ-amide folate adduct was synthesized; both negatively charged carboxylates in the glutamate tail were replaced with positively charged amines. The Ki for this adduct was ~9-fold higher than for folate. These various results indicate a balance between folate tail disorder, which helps the enzyme bind substrate while dynamics facilitates catalysis.},
doi = {10.1021/acs.biochem.5b00981},
journal = {Biochemistry},
issn = {0006-2960},
number = 1,
volume = 55,
place = {United States},
year = {2015},
month = {12}
}