Solution-state NMR investigations of triosephosphate isomerase active site loop motion: ligand release in relation to active site loop dynamics11Edited by P. E. Wright

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The highly efficient glycolytic enzyme, triosephosphate isomerase, is expected to differentially stabilize the proposed stable reaction species: ketone, aldehyde, and enediol(ate). The identity and steady-state populations of the chemical entities bound to triosephosphate isomerase have been probed by using solid-and solution-state NMR. The 13 C-enriched ketone substrate, dihydroxyacetone phosphate, was bound to the enzyme and characterized at steady state over a range of sample conditions. The ketone substrate was observed to be the major species over a temperature range from ؊60°C to 15°C. Thus, there is no suggestion that the enzyme preferentially stabilizes the reactive intermediate or the product. The predominance of dihydroxyacetone phosphate on the enzyme would support a mechanism in which the initial proton abstraction in the reaction from dihydroxyacetone phosphate to D-glyceraldehyde 3-phosphate is significantly slower than the subsequent chemical steps.enzymatic catalysis ͉ Michaelis complex ͉ solid-state NMR T he glycolytic enzyme triosephosphate isomerase (TIM) catalyzes isomerization of a ketone to an aldehyde, progressing via two successive proton transfers involving carbon acids and additional proton transfers for the attached oxygen atoms (Fig. 1). The precise path of the transferred proton and the identity of the reaction intermediates have been, by and large, deduced from the fate of isotopic labels (1, 2). The existence of a stable enediol(ate) intermediate has been inferred from the loss of an isotope label from the substrate during the reaction (1, 3); this reaction intermediate must exist with a sufficient lifetime to allow partial equilibration of the catalytic base with the surrounding solvent. Moreover, when the reaction takes place in tritiated water the solvent's tritium is incorporated into both the substrate and product, evidence that the hydrogen of the newly formed carbonhydrogen bond is mostly derived from the solvent (4, 5) and that exchange occurs from an enzyme bound intermediate. The stereochemistry of the reaction (2) and the orientation of the substrate in the Michaelis complex (6) strongly suggest that the reaction intermediate is a cis-enediol(ate). Based on a comprehensive study of isotope effects supplemented by extensive biophysical investigations of the reaction mechanism (7, 8), Albery and Knowles (9) constructed a kinetically detailed energy profile for the reversible interconversion of substrate, product, and one intermediate subject to isotope exchange with solvent ( Fig. 1).This now well known reaction energy profile illustrated for the first time many of the essential elements contributing to the high catalytic efficiency brought about by enzymes (10). For example, the free-energy profile illustrates the potential catalytic power of preferential stabilization of intermediates in a reaction. The high catalytic efficiency of TIM [almost 10 orders of magnitude compared with the reaction catalyzed by an acetate ion (11)] is thought to arise from matching the pK a va...

Section: Resultsmentioning

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Substrate product equilibrium on a reversible enzyme, triosephosphate isomerase

Rozovsky

McDermott

2007

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“…We have performed the experiments at 4°C, where the side reaction is minimized (28). Crystals of unligated yeast TIM [W90Y, W157F, 5Ј-fluorotryptophan at W168, a fully active mutant used in NMR studies (25,26)] were soaked in a solution containing DHAP. Prompt deterioration of these crystals was observed upon soaking, and the mosaicity of the crystal increased from 0.3 to 1.4°after soaking for 2 h. This degradation presumably occurs because of the previously reported conformational change for the flexible loop in the active site, as this loop is involved in crystal-packing contacts in the unligated enzyme (9).…”

Section: Resultsmentioning

confidence: 99%

“…Biochemical and genetic evidence suggests that the loop, and its precise sequence, is critical for function (42). Kinetic and spectroscopic measurements indicate that the time scale of loop motion matches that of product release and turnover, suggesting that the reaction intermediate is effectively protected from water throughout its lifetime (25,50). How is the loop opening coordinated with the completion of the chemical reaction?…”

Section: Resultsmentioning

confidence: 99%

Optimal alignment for enzymatic proton transfer: Structure of the Michaelis complex of triosephosphate isomerase at 1.2-Å resolution

Jogl

Rozovsky

McDermott

et al. 2002

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135

281

In enzyme catalysis, where exquisitely positioned functionality is the sine qua non, atomic coordinates for a Michaelis complex can provide powerful insights into activation of the substrate. We focus here on the initial proton transfer of the isomerization reaction catalyzed by triosephosphate isomerase and present the crystal structure of its Michaelis complex with the substrate dihydroxyacetone phosphate at near-atomic resolution. The active site is highly compact, with unusually short and bifurcated hydrogen bonds for both catalytic Glu-165 and His-95 residues. The carboxylate oxygen of the catalytic base Glu-165 is positioned in an unprecedented close interaction with the ketone and the ␣-hydroxy carbons of the substrate (C. . . O Ϸ 3.0 Å), which is optimal for the proton transfer involving these centers. The electrophile that polarizes the substrate, His-95, has close contacts to the substrate's O1 and O2 (N. . . O < 3.0 and 2.6 Å, respectively). The substrate is conformationally relaxed in the Michaelis complex: the phosphate group is out of the plane of the ketone group, and the hydroxy and ketone oxygen atoms are not in the cisoid configuration. The ammonium group of the electrophilic Lys-12 is within hydrogen-bonding distance of the substrate's ketone oxygen, the bridging oxygen, and a terminal phosphate's oxygen, suggesting a role for this residue in both catalysis and in controlling the flexibility of active-site loop.T riosephosphate isomerase (TIM) catalyzes the isomerization between dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (GAP). The uphill direction from DHAP to GAP is essential for optimal throughput in the glycolytic pathway. To accomplish this reaction, TIM extracts the pro-R hydrogen from the C1 carbon of DHAP and then stereo-specifically introduces a proton to the C2 carbon ( Fig. 1 a and b; ref. 1). Kinetic isotope effects and isotope ''washout'' experiments suggest that the reaction proceeds through a planar cis-enediol or enediolate intermediate of moderate stability (2, 3), and that the energetic landscape is characterized by several steps of competitive timescales (4). The crucial proton transfers between C1 and C2 atoms of the substrate are most likely carried out by a single base, the side chain carboxylate of Glu-165, whereas His-95 and Lys-12 probably facilitate the transfer of the hydroxyl proton from O1 to O2 (Fig. 1a; ref. 5). Structural studies revealed an ␣͞␤ fold, now known as the TIM-barrel (6-8), and elucidated the geometry of the catalytic residues at the active site and their interactions with the ligands (9).TIM is a textbook case in enzymatic enolization chemistry and has become the subject of landmark spectroscopic and computational studies elucidating the details of the mechanism, the protein motions relevant to chemistry, and the design principles that allow efficient and uphill proton transfer in enzyme active sites. Spectroscopic and mutation studies have focused on the polarization of the substrate by catalytic residues as well as on the...

“…Application of these methods to a number of enzymes has revealed motions of active site loops on the time scale of catalysis (33)(34)(35)(36). However, it has generally proved difficult to characterize structurally the excited state that is involved in the conformational averaging.…”

Section: Discussionmentioning

confidence: 99%

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

McElheny

Schnell²,

Lansing³

et al. 2005

154

229

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...