Dynamics of the Dihydrofolate Reductase-Folate Complex: Catalytic Sites and Regions Known To Undergo Conformational Change Exhibit Diverse Dynamical Features

Epstein, David; Benkovic, Stephen J.; Wright, Peter E.

doi:10.1021/bi00035a009

Cited by 161 publications

(162 citation statements)

References 62 publications

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“…Previous spin relaxation studies revealed large-amplitude picosecond-to-nanosecond time scale motions of the Met-20 loop in the occluded states of the enzyme that are abrogated upon loop closure (4,17,18). These studies also hinted at changes in microsecond-tomillisecond time scale motions that are potentially relevant to catalysis.…”

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confidence: 95%

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.

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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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mentioning

confidence: 95%

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.

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153

229

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“…Protein dynamics on the ps-ns and μs-ms timescales have been examined for a number of DHFR complexes by using NMR-based techniques (12)(13)(14)(15)(16)(17). The E:FOL and E:FOL: 5′,6′-dihydroNADPH (DHNADPH) complexes are both in the occluded conformation and display very similar ps-ns backbone dynamics that differ from those of the closed E:FOL:NADP þ complex (16).…”

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confidence: 99%

Millisecond timescale fluctuations in dihydrofolate reductase are exquisitely sensitive to the bound ligands

Boehr

McElheny

Dyson

et al. 2010

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

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171

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Enzyme catalysis can be described as progress over a multidimensional energy landscape where ensembles of interconverting conformational substates channel the enzyme through its catalytic cycle. We applied NMR relaxation dispersion to investigate the role of bound ligands in modulating the dynamics and energy landscape of Escherichia coli dihydrofolate reductase to obtain insights into the mechanism by which the enzyme efficiently samples functional conformations as it traverses its reaction pathway. Although the structural differences between the occluded substrate binary complexes and product ternary complexes are very small, there are substantial differences in protein dynamics. Backbone fluctuations on the μs-ms timescale in the cofactor binding cleft are similar for the substrate and product binary complexes, but fluctuations on this timescale in the active site loops are observed only for complexes with substrate or substrate analog and are not observed for the binary product complex. The dynamics in the substrate and product binary complexes are governed by quite different kinetic and thermodynamic parameters. Analogous dynamic differences in the E:THF:NADPH and E:THF:NADP þ product ternary complexes are difficult to rationalize from ground-state structures. For both of these complexes, the nicotinamide ring resides outside the active site pocket in the ground state. However, they differ in the structure, energetics, and dynamics of accessible higher energy substates where the nicotinamide ring transiently occupies the active site. Overall, our results suggest that dynamics in dihydrofolate reductase are exquisitely "tuned" for every intermediate in the catalytic cycle; structural fluctuations efficiently channel the enzyme through functionally relevant conformational space. catalysis | energy landscape | enzyme dynamics | NMR | relaxation

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“…To this day, only a small number of enzymes have been shown to rely on essential proximal and/or distal coupled residue motions for catalysis, among which dihydrofolate reductase (5)(6)(7)(8), cyclophilin A (9, 10), liver alcohol dehydrogenase (11)(12)(13)(14), triose-phosphate isomerase (15)(16)(17)(18)(19), and ribonuclease A (20 -22) remain some of the best characterized systems (for recent reviews see Refs. 23 and 24).…”

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confidence: 99%

NMR Investigation of Tyr105 Mutants in TEM-1 β-Lactamase

Doucet¹,

Savard

Pelletier

et al. 2007

Journal of Biological Chemistry

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The existence of coupled residue motions on various time scales in enzymes is now well accepted, and their detailed characterization has become an essential element in understanding the role of dynamics in catalysis. To this day, a handful of enzyme systems has been shown to rely on essential residue motions for catalysis, but the generality of such phenomena remains to be elucidated. Using NMR spectroscopy, we investigated the electronic and dynamic effects of several mutations at position 105 in TEM-1 ␤-lactamase, an enzyme responsible for antibiotic resistance. Even in absence of substrate, our results show that the number and magnitude of short and long range effects on 1 H-15 N chemical shifts are correlated with the catalytic efficiencies of the various Y105X mutants investigated. In addition, 15 N relaxation experiments on mutant Y105D show that several active-site residues of TEM-1 display significantly altered motions on both picosecond-nanosecond and microsecond-millisecond time scales despite many being far away from the site of mutation. The altered motions among various activesite residues in mutant Y105D may account for the observed decrease in catalytic efficiency, therefore suggesting that short and long range residue motions could play an important catalytic role in TEM-1 ␤-lactamase. These results support previous observations suggesting that internal motions play a role in promoting protein function. Enzymes are extremely efficient catalysts that can accelerate biochemical reactions up to a factor of 10 18 when compared with the same uncatalyzed reaction (1). Although considerable progress has been made in understanding enzyme catalysis over the past few years (2, 3), the detailed explanation of this large rate enhancement remains a significant challenge. Historically regarded as relatively static entities, increasing evidence now suggests that enzymes behave as dynamic machines and that motions on various time scales play important roles in promoting enzyme catalysis (4). To this day, only a small number of enzymes have been shown to rely on essential proximal and/or distal coupled residue motions for catalysis, among which dihydrofolate reductase (5-8), cyclophilin A (9, 10), liver alcohol dehydrogenase (11-14), triose-phosphate isomerase (15-19), and ribonuclease A (20 -22) remain some of the best characterized systems (for recent reviews see Refs. 23 and 24). Analogous behavior among other enzymes remains to be elucidated, although the confirmation of such phenomena in structurally and functionally unrelated protein families and folds suggests that this may be a widespread process (24). A general view of such correlation between structure, function, and dynamics in enzymes would greatly improve our current understanding of these powerful catalysts. In this study, we provide experimental evidence supporting the importance of active-site residue motions in the enzyme TEM-1 ␤-lactamase through the characterization of various Y105X mutants by NMR spectroscopy.We have previously investigated th...

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Dynamics of the Dihydrofolate Reductase-Folate Complex: Catalytic Sites and Regions Known To Undergo Conformational Change Exhibit Diverse Dynamical Features

Cited by 161 publications

References 62 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

Millisecond timescale fluctuations in dihydrofolate reductase are exquisitely sensitive to the bound ligands

NMR Investigation of Tyr105 Mutants in TEM-1 β-Lactamase

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