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PDBsum entry 5ccc
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Oxidoreductase
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PDB id
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5ccc
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References listed in PDB file
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Key reference
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Title
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Cofactor-Mediated conformational dynamics promote product release from escherichia coli dihydrofolate reductase via an allosteric pathway.
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Authors
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D.Oyen,
R.B.Fenwick,
R.L.Stanfield,
H.J.Dyson,
P.E.Wright.
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Ref.
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J Am Chem Soc, 2015,
137,
9459-9468.
[DOI no: ]
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PubMed id
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Abstract
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The enzyme dihydrofolate reductase (DHFR, E) from Escherichia coli is a paradigm
for the role of protein dynamics in enzyme catalysis. Previous studies have
shown that the enzyme progresses through the kinetic cycle by modulating the
dynamic conformational landscape in the presence of substrate dihydrofolate
(DHF), product tetrahydrofolate (THF), and cofactor (NADPH or NADP(+)). This
study focuses on the quantitative description of the relationship between
protein fluctuations and product release, the rate-limiting step of DHFR
catalysis. NMR relaxation dispersion measurements of millisecond time scale
motions for the E:THF:NADP(+) and E:THF:NADPH complexes of wild-type and the
Leu28Phe (L28F) point mutant reveal conformational exchange between an occluded
ground state and a low population of a closed state. The backbone structures of
the occluded ground states of the wild-type and mutant proteins are very
similar, but the rates of exchange with the closed excited states are very
different. Integrated analysis of relaxation dispersion data and THF
dissociation rates measured by stopped-flow spectroscopy shows that product
release can occur by two pathways. The intrinsic pathway consists of spontaneous
product dissociation and occurs for all THF-bound complexes of DHFR. The
allosteric pathway features cofactor-assisted product release from the closed
excited state and is utilized only in the E:THF:NADPH complexes. The L28F
mutation alters the partitioning between the pathways and results in increased
flux through the intrinsic pathway relative to the wild-type enzyme. This
repartitioning could represent a general mechanism to explain changes in product
release rates in other E. coli DHFR mutants.
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