Structure of the triosephosphate isomerase-phosphoglycolohydroxamate complex: an analog of the intermediate on the reaction pathway

Phosphoglucose isomerase (EC 5.3.1.9) catalyzes the second step in glycolysis, the reversible isomerization of D-glucose 6-phosphate to D-fructose 6-phosphate. The reaction mechanism involves acid-base catalysis with proton transfer and proceeds through a cis-enediol(ate) intermediate. 5-Phospho-D-arabinonohydroxamic acid (5PAH) is a synthetic small molecule that resembles the reaction intermediate, differing only in that it has a nitrogen atom in place of C1. Hence, 5PAH is the best inhibitor of the isomerization reaction reported to date with a Ki of 2 ؋ 10 ؊7 M. Here we report the crystal structure of rabbit phosphoglucose isomerase complexed with 5PAH at 1.9 Å resolution. The interaction of 5PAH with amino acid residues in the enzyme active site supports a model of the catalytic mechanism in which Glu-357 transfers a proton between C1 and C2 and Arg-272 helps stabilize the intermediate. It also suggests a mechanism for proton transfer between O1 and O2. P hosphoglucose isomerase (PGI; EC 5.3.1.9) is a cytosolic glycolytic enzyme that catalyzes the reversible isomerization of D-glucose 6-phosphate (G6P) to D-fructose 6-phosphate (F6P). In addition to isomerase activity, PGI has been shown to display anomerase activity between pyranose anomers of G6P (1), between furanose anomers of F6P (2), and between those of D-mannose 6-phosphate (3), as well as C-2-epimerase activity on G6P (4). It also has roles in protein glycosylation, gluconeogenesis, and the pentose phosphate pathway (5). This central role in the metabolism of phosphorylated sugars explains the strong impact of PGI deficiency in humans (6, 7), as well as the interest as a therapeutic target in parasite metabolism (8). The PGI polypeptide chain, or perhaps a variation thereof, also has extracellular roles as neuroleukin, autocrine motility factor, and differentiation and maturation mediator (9-12). These proteins promote antibody secretion by mononuclear cells, tumor cell motility, and differentiation of leukemia cells and a promyelocytic cell line (HL-60 cells), respectively. More recently, PGI has also been identified as the antigen involved in rheumatoid arthritis of a mouse line (13), as a specific inhibitor toward myofibril-bound serine proteinase (14), and as the surface antigen involved in sperm agglutination (15). Because very little is known about how PGI is precisely involved in these various biological processes, this moonlighting protein (16) has been the subject of intense investigations.Biochemical characterization of PGI began over 50 years ago and includes inhibitor studies (1,(17)(18)(19)(20)(21)(22)(23)(24)(25), labeling studies (26-32), solvent exchange (33, 34), mutagenesis (35,36), and pH profile studies (20,37). A proposed multistep catalytic mechanism includes a ring-opening step followed by an isomerization step. The isomerization step proceeds via general acid-base catalysis with proton transfer. In the G6P to F6P direction, abstraction of the proton from C2 by an active site amino acid residue yields a 1,2-cis-enediol(ate) in...

Section: Comparison Of Pgi͞5pah To Complexes Of Tim and Xyl With Analmentioning

confidence: 99%

mentioning

confidence: 99%

The crystal structure of rabbit phosphoglucose isomerase complexed with 5-phospho- d -arabinonohydroxamic acid

Arsenieva

Hardré

Salmon

et al. 2002

“…Alterations to the shape or charge of these amino acids should disrupt tertiary interactions. To test this prediction, the conserved residue positions in the aligned TIM genes were assigned to seven structural classes by examination of the yeast crystal structure (17). Because the (␤͞␣) 8 barrel is composed of a repeated structural motif, each structural class is represented by multiple residues.…”

Section: Mutability At Phylogenetically Hydrophobic and Polar Positionsmentioning

confidence: 99%

Reverse engineering the (β/α) ₈ barrel fold

Silverman

Balakrishnan

Harbury

2001

The (␤͞␣)8 barrel is the most commonly occurring fold among protein catalysts. To lay a groundwork for engineering novel barrel proteins, we investigated the amino acid sequence restrictions at 182 structural positions of the prototypical (␤͞␣)8 barrel enzyme triosephosphate isomerase. Using combinatorial mutagenesis and functional selection, we find that turn sequences, ␣-helix capping and stop motifs, and residues that pack the interface between ␤-strands and ␣-helices are highly mutable. Conversely, any mutation of residues in the central core of the ␤-barrel, ␤-strand stop motifs, and a single buried salt bridge between amino acids R189 and D227 substantially reduces catalytic activity. Four positions are effectively immutable: conservative single substitutions at these four positions prevent the mutant protein from complementing a triosephosphate isomerase knockout in Escherichia coli. At 142 of the 182 positions, mutation to at least one amino acid of a seven-letter amino acid alphabet produces a triosephosphate isomerase with wild-type activity. Consequently, it seems likely that (␤͞␣)8 barrel structures can be encoded with a subset of the 20 amino acids. Such simplification would greatly decrease the computational burden of (␤͞␣)8 barrel design.

“…5). Structural studies revealed an ␣͞␤ fold, now known as the TIM-barrel (6)(7)(8), and elucidated the geometry of the catalytic residues at the active site and their interactions with the ligands (9).…”

mentioning

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

134

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...