7 research outputs found
Temporally Overlapped but Uncoupled Motions in Dihydrofolate Reductase Catalysis
Temporal correlations between protein motions and enzymatic reactions
are often interpreted as evidence for catalytically important motions.
Using dihydrofolate reductase as a model system, we show that there
are many protein motions that temporally overlapped with the chemical
reaction, and yet they do not exhibit the same kinetic behaviors (KIE
and pH dependence) as the catalyzed chemical reaction. Thus, despite
the temporal correlation, these motions are not directly coupled to
the chemical transformation, and they might represent a different
part of the catalytic cycle or simply be the product of the intrinsic
flexibility of the protein
Kinetic Mechanism of Indole-3-glycerol Phosphate Synthase
The (βα)<sub>8</sub>-barrel enzyme indole-3-glycerol
phosphate synthase (IGPS) catalyzes the multistep transformation of
1-(<i>o</i>-carboxyphenylamino)-1-deoxyribulose 5-phosphate
(CdRP) into indole-3-glycerol phosphate (IGP) in tryptophan biosynthesis.
Mutagenesis data and crystal structure analysis of IGPS from <i>Sulfolobus solfataricus</i> (sIGPS) allowed for the formulation
of a plausible chemical mechanism of the reaction, and molecular dynamics
simulations suggested that flexibility of active site loops might
be important for catalysis. Here we developed a method that uses extrinsic
fluorophores attached to active site loops to connect the kinetic
mechanism of sIGPS to structure and conformational motions. Specifically,
we elucidated the kinetic mechanism of sIGPS and correlated individual
steps in the mechanism to conformational motions of flexible loops.
Pre-steady-state kinetic measurements of CdRP to IGP conversion monitoring
changes in intrinsic tryptophan and IGP fluorescence provided a minimal
three-step kinetic model in which fast substrate binding and chemical
transformation are followed by slow product release. The role of sIGPS
loop conformational motion during substrate binding and catalysis
was examined via variants that were covalently labeled with fluorescent
dyes at the N-terminal extension of the enzyme and mobile active site
loop β1α1. Analysis of kinetic data monitoring dye fluorescence
revealed a conformational change that follows substrate binding, suggesting
an induced-fit-type binding mechanism for the substrate CdRP. Global
fitting of all kinetic results obtained with wild-type sIGPS and the
labeled variants was best accommodated by a four-step kinetic model.
In this model, both the binding of CdRP and its on-enzyme conversion
to IGP are accompanied by conformational transitions. The liberation
of the product from the active site is the rate-limiting step of the
overall reaction. Our results confirm the importance of flexible active
loops for substrate binding and catalysis by sIGPS
SDS-PAGE gel of <i>Wb</i>DHFR protein after purification and concentration.
<p>Lane 1: SeeBlue Plus2 Pre-stained Protein Standard (Novex); Lane 2: Column flow through; Lane 3: Purified <i>Wb</i>DHFR (8.25 μg).</p
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CLUSTAL alignment between <i>Wb</i>, <i>Bm</i> and mouse DHFRs.
<p>The eight amino acid differences between <i>Wb</i> and <i>Bm</i>DHFR are marked with #. The 13 amino acid region missing from the Uniprot entry and designed into the gene construct based on homology to the <i>Bm</i>DHFR is shown in bold type and underlined. The *s denote identical residues conserved among the three sequences.</p
IC<sub>50</sub> values for compounds tested against <i>Bm</i>DHFR and <i>Wb</i>DHFR.
<p>IC<sub>50</sub> values for compounds tested against <i>Bm</i>DHFR and <i>Wb</i>DHFR.</p
Representative Michaelis-Menten curve.
<p>The conditions in the experimental wells (200 μL) were 100 μM NADPH, 12.4 nM <i>Wb</i>DHFR in 1 X MTEN buffer at pH 6.0 with DHF concentrations ranging from 0 to 195 μM. The Michaelis-Menten equation was fitted to the data using KaleidaGraph. The Michaelis-Menten constant for DHF was determined by averaging values from fitting three separate data sets and found to be 3.7 ± 2.0 μM (S.D.).</p