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

Abstract

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...

RETRACTED: Local Encoding of Computationally Designed Enzyme Activity

“…Comparison of the high-resolution X-ray structure of yeast TIM 20 , and the NovoTIM molecular model, suggests some obvious defects in the design (Fig. 1c-d).…”

“…In particular, the orientation of the active glutamate relative to the abstracted proton is non-ideal (distance too long, suboptimal alignment), whereas it is highly optimized in the yeast enzyme. 20 Although highresolution structures of catalytic antibodies have revealed excellent stereochemical complementarity between the antibody and the hapten6, sub-optimal encoding in catalytic antibodies may arise because the hapten is an imperfect surrogate for the transition state6 , 35, or because of difficulties in directing the immune response to generate sequences that place desired chemical functional groups as well as binding activity in the CDRs 6 . The limit of local encoding therefore remains an unresolved issue for both design strategies as implemented thus far.…”

“…The substrate conformations are also slightly different. In the modeled design the phosphate is kept in plane with the carbons thereby applying a putative stereoelectronic effect to prevent β-elimination of the phosphate 9 ; in the high-resolution X-ray structure of the wildtype enzyme, the phosphate is somewhat out of plane 20 .…”

Oxyanion Hole-stabilized Stereospecific Isomerization in Ribose-5-phosphate Isomerase (Rpi)

“…Structural studies on eukaryotic TIM complexed with inhibitors such as phosphoglycolohydroxamate (PGH) and 2-(N-formyl-Nhydroxy)-aminoethylphosphate (IPP) and the substrate dihydroxyacetone phosphate (DHAP) have supported the suggestion that the catalytic mechanism in the conversion of DHAP and glyceraldehyde-3-phosphate is a C1 and C2 proton transfer via a cis-enediol(ate) intermediate (26,29,30). Glu-165 in TIM is proposed to be the catalytic base, whereas His-95 and Lys-12 probably facilitate proton transfer between O1 and O2.…”

Enzymes with lid-gated active sites must operate by an induced fit mechanism instead of conformational selection

“…This results in the occlusion of the active site from the bulk solvent (20)(21)(22)(23)(24). These conformational changes, often required to preclude solvent from competing with the catalzyed reaction (25,26), also preclude substrate binding directly to this enzyme form.…”