High resolution crystal structures of triosephosphate isomerase complexed with its suicide inhibitors: the conformational flexibility of the catalytic glutamate in its closed, liganded active site

Abstract

The key residue of the active site of triosephosphate isomerase (TIM) is the catalytic glutamate, which is proposed to be important (i) as a catalytic base, for initiating the reaction, as well as (ii) for the subsequent proton shuttling steps. The structural properties of this glutamate in the liganded complex have been investigated by studying the high resolution crystal structures of typanosomal TIM, complexed with three suicide inhibitors: (S)-glycidol phosphate ((S)-GOP, at 0.99 Å resolution), (R)-glycidol phosphate, ((R)-GOP, at 1.08 Å resolution), and bromohydroxyacetone phosphate (BHAP, at 1.97 Å resolution). The structures show that in the (S)-GOP active site this catalytic glutamate is in the well characterized, competent conformation. However, an unusual side chain conformation is observed in the (R)-GOP and BHAP complexes. In addition, Glu97, salt bridged to the catalytic lysine in the competent active site, adopts an unusual side chain conformation in these two latter complexes. The higher chemical reactivity of (S)-GOP compared with (R)-GOP, as known from solution studies, can be understood: the structures indicate that in the case of (S)-GOP, Glu167 can attack the terminal carbon of the epoxide in a stereoelectronically favored, nearly linear OCO arrangement, but this is not possible for the (R)-GOP isomer. These structures confirm the previously proposed conformational flexibility of the catalytic glutamate in its closed, liganded state. The importance of this conformational flexibility for the proton shuttling steps in the TIM catalytic cycle, which is apparently achieved by a sliding motion of the side chain carboxylate group above the enediolate plane, is also discussed.

Figure 1

(A) The TIM reaction. Glu167 is the catalytic base which generates the initial enediolate intermediate from the substrate, DHAP. Several additional proton shuttling steps complete the reaction cycle to the product, d-GAP., (B) The covalent structures and nomenclatures of the (R)-GOP, (S)-GOP, and BHAP suicide inhibitors. The systematic name of GOP is glycidyl phosphate. The (S)-GOP enantiomer corresponds to d-GOP and has the same chirality as the substrate d-GAP. The (R)-GOP enantiomer corresponds to l-GOP. The covalent structures of the transition state analog 2PG, and the reaction intermediate analogs PGH and IPP are also shown. PGH is a mimic of the ketoform of the substrate and IPP is a mimic of the aldehyde form of the substrate. (C) The covalent modification reaction of (S)-GOP. (D) The covalent modification reaction of BHAP. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2

The competent active site at the dimer interface of the TIM-PGH complex (2VXN). The dotted lines mark the hydrogen bonding network between O2(PGH), NZ(Lys13), Glu97, Thr75*, and Asn11, including the water mediated hydrogen bond between NZ(Lys13) and the phosphate moiety. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 3

The omit map of (S)-GOP, calculated after omit refinement at 0.99 Å resolution. These omit calculations were done with a model obtained from the final (S)-GOP structure by deleting the inhibitor atoms as well as the Glu167 side chain atoms. The atoms of both the major form ((S)-GOP) and the minor form ((R)-GOP) of GOP as well as the Glu167 atoms of the final (S)-GOP structure are shown. The map is contoured at 2.8σ. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 4

The comparison of the active site geometry of unliganded TIM (purple, 5TIM) and the active sites complexed with IPP (cyan, 1IF2), 2PG (magenta, 1N55), DHAP (green, 1NEY) and PGH (yellow, 2VXN). The comparison highlights the conformational flexibility of Glu167 in the competent active site.

Figure 5

The structures of the (S)-GOP complex (blue), (R)-GOP complex (magenta), and the BHAP-complex (green), compared with the PGH complex (yellow, 2VXN). Thr75*, Glu97, Asn11, Lys13, His95, and Glu167 are also shown. A water molecule (O, orange) replaces the Glu97 side chain in the (R)-GOP and BHAP structures.

Figure 6

The structure of (R)-GOP (magenta) compared with the IPP complex (cyan, 1IF2) and the PGH complex (yellow, 2VXN), visualizing the hydrophobic cage of the Glu167 side chain, shaped by Cys126, Ala165, Ile172, Gly211, and Leu232. The Glu167 side chain is near Cys126, Ala165 in the PGH complex, mimicking the enzyme-DHAP complex, and near Gly211 (Loop-7), Ile172 (Loop-6) in the IPP complex, mimicking the enzyme–d-GAP complex.