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)₈-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

IC₅₀ values for compounds tested against <i>Bm</i>DHFR and <i>Wb</i>DHFR.

<p>IC₅₀ 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