The adaptability of the active site of trypanosomal triosephosphate isomerase as observed in the crystal structures of three different complexes

Noble, M.E.M.; Wierenga, Rik K.; Lambeir, Anne‐Marie; Opperdoes, Frederik; Thunnissen, A.M.W.H.; Kalk, Kornelis; Groendijk, H.; Hol, W.G.J.

doi:10.1002/prot.340100106

Cited by 82 publications

(72 citation statements)

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“…This asymmetry has been observed earlier in trypanosomal TIM complexed with various ligands but appears to be a result of crystallographic contacts (52,53). In PfTIM, asymmetry seems to be an intrinsic, although puzzling property not related to crystallographic packing.…”

Section: Interpretation Of the Fragmented Ligand Electron Density Insupporting

confidence: 60%

Structure of Plasmodium falciparum Triose-phosphate Isomerase-2-Phosphoglycerate Complex at 1.1-Å Resolution

Parthasarathy

Kandiah

Balaram

et al. 2003

Journal of Biological Chemistry

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Triose-phosphate isomerase, a key enzyme of the glycolytic pathway, catalyzes the isomerization of dihydroxy acetone phosphate and glyceraldehyde 3-phosphate. In this communication we report the crystal structure of Plasmodium falciparum triose-phosphate isomerase complexed to the inhibitor 2-phosphoglycerate at 1.1-Å resolution. The crystallographic asymmetric unit contains a dimeric molecule. The inhibitor bound to one of the subunits in which the flexible catalytic loop 6 is in the open conformation has been cleaved into two fragments presumably due to radiation damage. The cleavage products have been tentatively identified as 2-oxoglycerate and meta-phosphate. The intact 2-phosphoglycerate bound to the active site of the other subunit has been observed in two different orientations. The active site loop in this subunit is in both open and "closed" conformations, although the open form is predominant. Concomitant with the loop closure, Phe-96, Leu-167, and residues 208 -211 (YGGS) are also observed in dual conformations in the B-subunit. Detailed comparison of the active-site geometry in the present case to the Saccharomyces cerevisiae triose-phosphate isomerase-dihydroxy acetone phosphate and Leishmania mexicana triose-phosphate isomerase-phosphoglycolate complexes, which have also been determined at atomic resolution, shows that certain interactions are common to the three structures, although 2-phosphoglycerate is neither a substrate nor a transition state analogue. Triose-phosphate isomerase (TIM)1 is a ubiquitous glycolytic enzyme that catalyzes the isomerization of dihydroxy acetone phosphate (DHAP) and glyceraldehyde 3-phosphate through the intermediate formation of cis-enediol(ate(s)). Commenting on the catalytic efficiency of TIM, Knowles (1) states "This enzyme (TIM) appears to have arrived at the end of its evolutionary development as a catalyst." It has been argued that the evolutionary pressure for TIM to be a perfect catalyst has been intense, since for "flight or fight" there is an instant requirement for muscle ATP. The catalytic reaction of TIM is chemically simple and involves intermolecular protonation and deprotonation between the enzyme and substrate. The enzyme achieves the proton transfers with the help of certain elements as catalytic tools, a 10-residue loop that closes over the active site and stabilizes the reaction intermediate and a catalytic base and an acid to perform the enolization steps (1). It is believed that catalysis is a result of stronger binding of the enzyme to its transition state than to the initial enzyme-substrate complex (2). Although various schemes for this reaction have been proposed (for a review of the schemes, see Cui and Karplus (3)), the most frequently discussed mechanism is the one that involves Glu-165 as the catalytic base in the first proton transfer and His-95 as the catalytic acid for the rest of the reaction (3) (Fig. 1a). Despite numerous experimental (4 -9) and theoretical studies (10, 11), the precise mechanism of the multi-step reaction cataly...

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Section: Interpretation Of the Fragmented Ligand Electron Density Insupporting

confidence: 60%

Structure of Plasmodium falciparum Triose-phosphate Isomerase-2-Phosphoglycerate Complex at 1.1-Å Resolution

Parthasarathy

Kandiah

Balaram

et al. 2003

Journal of Biological Chemistry

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“…This lysine has the same "unallowed" 6, $ combination in other TIM structures elucidated so far, including the higher resolution structures of trypanosoma1 as well as yeast TIMs Wierenga et al, 1992;Noble et al, 1993;Mande et al, 1994). In the B. stearothermophilus structure, all atoms of this residue have low temperature factors.…”

Section: Quality Of the Structurementioning

confidence: 74%

“…The loop formed by residues 168-178, called the "flexible loop," closes the active site when the substrate binds (Knowles, 1991). Several substrate analogue inhibitor structures have already been reported showing three different conformations of the flexible loop: "open" when there is no ligand at the active site (Noble, 1992), "partially closed" when sulfate is bound (Wierenga et al, 1991a), and "closed" when substrate analogue inhibitors are bound Noble et al, 1991aNoble et al, , 1991b. A unique case has been reported in which N-hydroxy-4-phosphono-butanamide, an inhibitor that is one carbon atom longer than the substrate, binds with the open conformation of the loop (Verlinde et al, 1 992).…”

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confidence: 99%

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Crystal structure of recombinant triosephosphate isomerase from bacillus stearothermophilus. An analysis of potential thermostability factors in six isomerases with known three‐dimensional structures points to the importance of hydrophobic interactions

et al. 1995

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The structure of the thermostable triosephosphate isomerase (TIM) from Bacillus stearothermophilus complexed with the competitive inhibitor 2-phosphoglycolate was determined by X-ray crystallography to a resolution of 2.8 A. The structure was solved by molecular replacement using XPLOR. Twofold averaging and solvent flattening was applied to improve the quality of the map. Active sites in both the subunits are occupied by the inhibitor and the flexible loop adopts the "closed" conformation in either subunit. The crystallographic R-factor is 17.6% with good geometry. The two subunits have an RMS deviation of 0.29 A for 248 C" atoms and have average temperature factors of 18.9 and 15.9 A*, respectively. In both subunits, the active site Lys 10 adopts an unusual $,$ combination.A comparison between the six known thermophilic and mesophilic TIM structures was conducted in order to understand the higher stability of B. stearothermophilus TIM. Although the ratio Arg/(Arg+Lys) is higher in B. stearotherrnophilus TIM, the structure comparisons do not directly correlate this higher ratio to the better stability of the B. stearothermophilus enzyme. A higher number of prolines contributes to the higher stability of B. stearothermophilus TIM. Analysis of the known TIM sequences points out that the replacement of a structurally crucial asparagine by a histidine at the interface of monomers, thus avoiding the risk of deamidation and thereby introducing a negative charge at the interface, may be one of the factors for adaptability at higher temperatures in the TIM family. Analysis of buried cavities and the areas lining these cavities also contributes to the greater thermal stability of the B. stearotherrnophilus enzyme. However, the most outstanding result of the structure comparisons appears to point to the hydrophobic stabilization of dimer formation by burying the largest amount of hydrophobic surface area in B. stearothermophilus TIM compared to all five other known TIM structures.

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“…The side chain of Glu-165 swings by about 2 Å upon closure of the adjacent active-site loop, allowing Glu-165 to contact the bound ligand. Moreover, the placement of this side chain seemed to depend on the selection of active-site ligand (39,40). In the Michaelis complex, one oxygen of the carboxylate interacts almost symmetrically with C1 and C2 (Fig.…”

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

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

134

281

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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...

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The adaptability of the active site of trypanosomal triosephosphate isomerase as observed in the crystal structures of three different complexes

Cited by 82 publications

References 40 publications

Structure of Plasmodium falciparum Triose-phosphate Isomerase-2-Phosphoglycerate Complex at 1.1-Å Resolution

Structure of Plasmodium falciparum Triose-phosphate Isomerase-2-Phosphoglycerate Complex at 1.1-Å Resolution

Crystal structure of recombinant triosephosphate isomerase from bacillus stearothermophilus. An analysis of potential thermostability factors in six isomerases with known three‐dimensional structures points to the importance of hydrophobic interactions

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

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