Interloop Contacts Modulate Ligand Cycling during Catalysis by<i>Escherichia coli</i>Dihydrofolate Reductase

Miller, Grover P.; Wahnon, Daphne; Benkovic, Stephen J.

doi:10.1021/bi001608n

Cited by 57 publications

(93 citation statements)

References 28 publications

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“…Previous kinetic studies of DHFR have revealed the important role played by the Met-20, FG, and GH loops in stabilization of the transition state and in control of substrate, product, and cofactor binding affinity and turnover (12)(13)(14). Our present relaxation dispersion measurements provide new insights into the mechanism by which millisecond time scale fluctuations of the active site loops, modulated by ligand-dependent changes in the relative stabilities of the major Met-20 loop conformations, are coupled to progression through the catalytic cycle.…”

Section: Discussionmentioning

confidence: 99%

“…Kinetic analysis of Met-20, FG, and GH loop mutants has confirmed their functional role in catalysis and in binding and release of cofactor, substrate, and products (12)(13)(14). The active site loops must cycle between the closed and occluded conformations at two steps in the reaction pathway, immediately after hydride transfer and again after product release; mutations that destabilize the closed conformation impair hydride transfer (13).…”

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confidence: 92%

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Defining the role of active-site loop fluctuations in dihydrofolate reductase catalysis

McElheny

Schnell²,

Lansing³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

154

229

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Dynamic processes are implicit in the catalytic function of all enzymes. To obtain insights into the relationship between the dynamics and thermodynamics of protein fluctuations and catalysis, we have measured millisecond time scale motions in the enzyme dihydrofolate reductase using NMR relaxation methods. Studies of a ternary complex formed from the substrate analog folate and oxidized NADP ؉ cofactor revealed conformational exchange between a ground state, in which the active site loops adopt a closed conformation, and a weakly populated (4.2% at 30°C) excited state with the loops in the occluded conformation. Fluctuations between these states, which involve motions of the nicotinamide ring of the cofactor into and out of the active site, occur on a time scale that is directly relevant to the structural transitions involved in progression through the catalytic cycle.enzyme catalysis ͉ hydride transfer ͉ NMR relaxation P roteins are dynamic molecular machines, and conformational fluctuations on a wide range of time scales are intimately associated with protein function. It has long been recognized that dynamic processes play an important role in the catalytic function of enzymes (1, 2). Protein motion is implicated in events such as binding of substrate or cofactor, allosteric regulation, and product release, and the catalyzed reaction itself is inherently dynamic, with changes in atomic positions occurring along the reaction coordinate (3). Despite mounting experimental evidence that the active sites of enzymes are inherently flexible (4-6), a detailed understanding of the relationship between the dynamics and thermodynamics of protein fluctuations and the catalytic process is currently lacking.A number of experimental and theoretical investigations have suggested that protein motions play an important role in catalysis by the enzyme dihydrofolate reductase (DHFR) (see refs. 7 and 8 for recent reviews). DHFR catalyzes the reduction of 7,8-dihydrofolate (DHF) through stereo-specific hydride transfer from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) cofactor. The enzyme is essential for tetrahydrofolate (THF) biosynthesis, plays a central role in promoting cell growth and proliferation, and is the target of several anticancer and antibiotic drugs. Escherichia coli DHFR has been subjected to extensive kinetic and structural studies that have defined the complete kinetic mechanism (9) and the structural transitions involved in the catalytic cycle (10, 11). During the reaction cycle (Fig. 1), the enzyme progresses through conformations in which the active site loops are in a closed state, in the holoenzyme and the Michaelis complex, and a series of product complexes in which the loops adopt an occluded conformation. In the closed state, the Met-20 loop (residues 9-24) packs against the nicotinamide ring of the cofactor and seals the active site (10). In the occluded state, Met-16 and Glu-17 in the Met-20 loop project into the active site and sterically occlude the binding site for the nicotinamide-r...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 92%

Defining the role of active-site loop fluctuations in dihydrofolate reductase catalysis

McElheny

Schnell²,

Lansing³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

154

229

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“…E. coli BL21(DE3) cells containing the plasmid pET27b-bioseq-N18C-C85A-C152S-DHFR were grown at 37°C in NCZYM medium containing 50 mg͞liter kanamycin to an absorbance of Ϸ0.5 at 600 nm and then induced at 37°C for 5 h with 0.4 mM isopropyl-␤-D-thiogalactopyranoside. After harvesting, the bioseq-N18C-C85A-C152S-DHFR protein was purified by using established procedures involving methotrexate affinity and DE-52 anionexchange chromatography (6). Twenty milligrams of the purified DHFR (Ϸ50 M in 20 ml) was biotinylated in vitro at 25°C for 24 h in 50 mM potassium phosphate, pH 7.2, containing 1 mM DTT, 4 mM biotin, 5 mM ATP, and 0.5 mg of biotin ligase (BirA-H 6 ).…”

Section: Experimental Methodsmentioning

confidence: 99%

“…Kinetic parameters of the labeled enzyme were determined by previously established procedures (2,6). All ensemble thermodynamic and kinetic measurements were carried out at 25°C in MTEN buffer (50 mM Mes͞25 mM Tris͞25 mM ethanolamine͞100 mM sodium chloride), pH 7.0.…”

Section: Experimental Methodsmentioning

confidence: 99%

Interaction of dihydrofolate reductase with methotrexate: Ensemble and single-molecule kinetics

Rajagopalan

Zhang

McCourt

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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276

216

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The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule was placed on a protein loop that closes over methotrexate when binding occurs, thus causing a quenching of the fluorescence. The biotin was used to attach the enzyme in an active form to a glass surface for single-molecule studies. The equilibrium dissociation constant for the binding of methotrexate to the enzyme is 9.5 nM. The stopped-flow studies revealed that methotrexate binds to two different conformations of the enzyme, and the association and dissociation rate constants were determined. The single-molecule investigation revealed a conformational change in the enzyme-methotrexate complex that was not observed in the stopped-flow studies. The ensemble averaged rate constants for this conformation change in both directions is about 2-4 s ؊1 and is attributed to the opening and closing of the enzyme loop over the bound methotrexate. Thus the mechanism of methotrexate binding to DHFR involves multiple steps and protein conformational changes.enzyme mechanisms ͉ protein dynamics ͉ fluorescence microscopy

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“…10 Indeed, in recent years much debate has emerged regarding the role of protein dynamics in enzyme catalysis in general 5,11,12 and in DHFR, in particular. 6,[13][14][15][16][17][18] How protein dynamics might contribute to catalysis ranges from conformational gating, where conformational changes might even be rate limiting, 19,20 as is thought to be the case with DHFR, [21][22][23] to a much more speculative direct coupling of specific protein motions to the reaction coordinate, perhaps similar to the strong and selective Fermi resonance between the stretching and bending motions of C-H moieties. [24][25][26] Unfortunately, the study of protein electrostatics and dynamics has been limited by the challenges associated with the direct characterization of specific protein bonds, including their environment and motion.…”

Section: Introductionmentioning

confidence: 99%

Carbon−Deuterium Bonds as Probes of Dihydrofolate Reductase

2008

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Much effort has been directed towards understanding the contributions of electrostatics and dynamics to protein function and especially to enzyme catalysis. Unfortunately, these studies have been limited by the absence of direct experimental probes. We have been developing the use of carbon-deuterium bonds as probes of proteins and now report the application of the technique to the enzyme dihydrofolate reductase, which catalyzes a hydride transfer and has served as a paradigm for biological catalysis. We observe that the stretching absorption frequency of (methyl-d 3 ) methionine carbon-deuterium bonds show an approximately linear dependence on solvent dielectric. Solvent and computational studies support the empirical interpretation of the stretching frequency in terms of local polarity. To begin to explore the use of this technique to study enzyme function and mechanism, we report a preliminary analysis of (methyl-d 3 ) methionine residues within dihydrofolate reductase. Specifically, we characterize the IR absorptions at Met16 and Met20, within the catalytically important Met20 loop, and Met42, which is located within the hydrophobic core of the enzyme. The results confirm the sensitivity of the carbon-deuterium bonds to their local protein environment, demonstrate that dihydrofolate reductase is electrostatically and dynamically heterogeneous, and lay the foundation for the direct characterization protein electrostatics and dynamics, and potentially, their contribution to catalysis.

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Interloop Contacts Modulate Ligand Cycling during Catalysis byEscherichia coliDihydrofolate Reductase

Cited by 57 publications

References 28 publications

Defining the role of active-site loop fluctuations in dihydrofolate reductase catalysis

Defining the role of active-site loop fluctuations in dihydrofolate reductase catalysis

Interaction of dihydrofolate reductase with methotrexate: Ensemble and single-molecule kinetics

Carbon−Deuterium Bonds as Probes of Dihydrofolate Reductase

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