Dynamics of a flexible loop in dihydrofolate reductase from Escherichia coli and its implication for catalysis

Apo-dihydrofolate reductase from Escherichia coli samples two distinct environments slowly on the NMR time scale at room temperature. Several assigned resonances belong to residues in, or proximal to, a loop (loop I) which is comprised of residues 9-24. This exchange process was altered (either remo...

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Bibliographic Details
Published in:Biochemistry (Easton) 1994-01, Vol.33 (2), p.439-442
Main Authors: Falzone, Christopher J, Wright, Peter E, Benkovic, Stephen J
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Catalysis
Conformational dynamics in molecular biology
Crystallization
Crystallography, X-Ray
Enzymes and enzyme inhibitors
Escherichia coli
Escherichia coli - enzymology
Folic Acid - metabolism
Fundamental and applied biological sciences. Psychology
Magnetic Resonance Spectroscopy
Methotrexate - metabolism
Molecular biophysics
Molecular Structure
NADP - metabolism
Oxidoreductases
Protein Conformation
Tetrahydrofolate Dehydrogenase - chemistry
Tetrahydrofolate Dehydrogenase - metabolism
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Summary:Apo-dihydrofolate reductase from Escherichia coli samples two distinct environments slowly on the NMR time scale at room temperature. Several assigned resonances belong to residues in, or proximal to, a loop (loop I) which is comprised of residues 9-24. This exchange process was altered (either removed or made fast on the NMR time scale) by deleting three hairpin turn forming residues from the loop and filling the gap with a single glycine [Li, L., Falzone, C. J., Wright, P. E., & Benkovic, S. J. (1992) Biochemistry 31, 7826-7833]. An approximate value of 35 s-1 for the exchange rate associated with loop I in apo-DHFR was obtained in two-dimensional nuclear Overhauser spectra by analyzing the time dependence of the cross-peak volume for N epsilon H of Trp-22, a residue which is located in this loop and which has resolved cross-peaks. Owing to the critical role that this loop plays in catalysis, the correspondence between this rate of conformational exchange and off-rates for tetrahydrofolate and the reduced nicotinamide cofactor from product and substrate complexes suggests that loop movement may be a limiting factor in substrate turnover.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00168a007