Cofactor-Mediated
Conformational Dynamics Promote
Product Release From Escherichia coli Dihydrofolate Reductase via an Allosteric Pathway
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Abstract
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<sup>+</sup>). 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<sup>+</sup> 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