Triose Phosphate Isomerase: Difference between revisions

TPI catalyzes the transfer of a hydrogen atom from carbon 1 to carbon 2, an intramolecular oxidation-reduction reaction [[Image:TPI 2D mechanism2.png|right|thumb|400px| '''Isomerization reaction catalyzed by TPI''']]. This isomerization of a ketose to an aldose proceeds through an ''cis''-enediol(ate) intermediate. This isomerization proceeds without any cofactors and the enzyme confers a 10⁹ rate enhancement relative to the nonenzymatic reaction involving a chemical base (acetate ion).<ref>PMID:2043623</ref>.  

TPI carries out the isomerization reaction through an acid-base-mediated mechanism involving <scene name='Triose_Phosphate_Isomerase/Three_catalytic_residues/3'>three catalytic residues</scene>.  First the DHAP or GAP subtrate is initially attracted to the enzyme active site through electrostatic interactions between the negatively charged substrate phosphate group and the positively charged <scene name='Triose_Phosphate_Isomerase/Lys12_shaded/1'>Lys12</scene>, with the resulting interaction stabilizing the substrate. According to the "classic" mechanism, <scene name='Triose_Phosphate_Isomerase/Glu165/3'>Glu165</scene> plays the role of the general base catalyst by abstracting a proton from the pro(''R'') position of carbon 1 of DHAP or the C-2 proton of GAP. However, the carboxylate group of Glutamate 165 alone does not possess the basicity to abstract a proton and requires <scene name='Triose_Phosphate_Isomerase/His95/6'>His95</scene>, the general acid, to donate a proton to stabilize the negative charge building up on C-2 carbonyl oxygen, effectively stabilizing the planar endediol(ate) intermediate,.  Lys12 and Asn11 also function to stabilize the negative charge which builds up on this intermediate. At this point in the mechanism, Glutamate 165 acts as a general acid by donating its proton to the neighboring C-2, while Histidine 95 now acts as a general base by abstracting a proton from the hydroxyl group of C-1. The final step in the reaction is the formation of the GAP isomer product while glutamate and histidine are returned to their original forms, regenerating the enzyme.  In studies using tritium labeled DHAP, Knowles observed only ~ 6% intramolecular transfer of the ³H label to the GAP product.  In explaining this result, Knowles argued that the hydrogen bound to the Glu165 was in equilibrium with those in bulk solvent.  Additionally, the reaction mechanism of the methylglyoxal forming enzyme methylglyoxal synthase (MGS) is believed to be similar to that of triosephosphate isomerase. Both enzymes utilize DHAP to form an enediol(ate) phosphate intermediate as the first step of their reaction pathways; however, the second catalytic step in the MGS reaction pathway features the elimination of phosphate and collapse of the enediol(ate) to form methylglyoxal rather then reprotonation to form the isomer glyceraldehyde 3-phosphate as seen in TPI.<ref>PMID:10368300</ref>

The formation and stabilization of the enediol(ate) intermediate has been a source of great discussion amongst scientists in the field. This is due to the fact that the formation of the enediol(ate) intermediate presents a large thermodynamic barrier involving the abstraction of the α-proton from the carbon acid substrate, an unfavorable process due to the lack of acidity and high p''K''a of the C1 proton of the substrate. The "classic" mechanism (shown above) put forth by Knowles and co-workers stated that the kinetic barrier is overcome by the concerted deprotonation of the C1 and the protonation of the C2 carbonyl oxygen which would effectively allow for equivalent free energies for each species shown shown, thus promoting an equilibrium constant near unity.<ref>PMID:9398185</ref> This concept of preferential binding arising from matching of the reaction energy barriers is a common theme of enzyme catalysis <ref>PMID:17287353</ref>.

[[Image:TPIfreeenergy2png.png|left|thumb|300px| '''Free-Energy Profile for the Reaction Catalyzed by Triosephosphate Isomerase''']]     

More recently a series of NMR experiments carried out by Mildvan and co-workers have shed light onto an alternative "Criss-cross" mechanism involving a LBHB between the catalytic Glu165 and the O1 oxygen of the substrate. This mechanism stipulates the His95 side chain does not directly transfer protons, this rather being accomplished entirely by Glu165.  Support for this mechanism was provided by Richard and coworkers who carried tritium labeling experiments demonstrating a significant amount of intramolecular transfer (49%) of the ¹H label from substrate (DHAP) to product (GAP)<ref>PMID:   15709774</ref>.  Using phosphoglycolohydroxamate (PGH), a mimic of the enediol(ate) intermediate, a 14.9 ppm chemical shift (6 ppm downfield) as well as a deuterium fractionation factor of 0.38 was observed with the TIM-PGH complex, corresponding to a highly deshielded proton involved in a LBHB between Glu165 and the hydroxamate oxygen of PGH. Conversely, the same NMR study found an additional hydrogen bond between the N-ε proton of His95 and the carbonyl oxygen of PGH; however, its chemical shift of 13.5 (0.4 ppm downfield from free enzyme) and fractionation factor of 0.71 indicated this was a strong H-bond, but not a LBHB.<ref>PMID:9748211</ref>.   

Although a highly studied enzyme, there are relatively few effective inhibitors of TPI.  From a pharmaceutical perspective, if TPI structures differ greatly between humans and microorganisms such as ''Plasmodium'' or ''Trypanosoma'', whose growth rely heavily or entirely on glycolysis, inhibition may be a strong therapeutic target.<ref>PMID:15911278</ref> Two irreversible inhibitors, halo-acetone phosphate and glycidol phosphate (1,2-epoxypropanol-3-P), act by labeling active site residues. Early biochemical studies involving glycidol phosphate have revealed the labeled residue to be the active site glutamate. There are several weak reversible inhibitors of TPI including 3-Phosphoglycerate, glycerol phosphate and phosphoenol pyruvate, with ''K''_i values ranging from 0.2-1.3 mM.<ref>PMID:15911278</ref>  Additionally, several transition state analogues have been used to study the mechanism of TPI, including phosphoglycolohydroxamate(PGH)(''K''_i = 6-14 μM) and the phosphoglycolic acid (PGA)(''K''_i = 3 μM) and 2(''N''-formyl-''N''-hydroxy)aminoethyl phosphonate (IPP) <ref>PMID:15911278</ref>. PGA (also called 2PG) believed to bind TPI as a trianion, undergoes tight active site binding through electrostatic interactions with both the neutral His95 and protonated Glu165 side chains. PGH (binding in the ''cis'' conformation) and IPP function by mimicking structural features of the cognate DHAP and GAP substrates, respectively<ref>PMID:12522213</ref>. Specifically, PGH effectively mimics the planar enediol(ate)intermediate.

As mentioned earlier, TPI is considered a catalytically perfect enzyme and accomplishes this largely due to its ability to suppress or prevent undesired side reactions such as the decomposition of the enediol intermediate into methyl glyoxal and orthophosphate, a process which is 100 fold faster in solution than the desired isomerization. TPI is able to prevent this undesired reaction by trapping and stabilizing the charged endiol(ate) intermediate in the active site through the use of a flexible 11 residue Ω loop referred to as <scene name='Triose_Phosphate_Isomerase/Morph_tpi/9'>Loop 6</scene> containing residues 168-178<ref>PMID:2402636</ref>, residue numbers variable with regards to species. Loop 6 can be further divided into a 3-residue N-terminal hinge, a rigid hydrophobic lid spanning 5-residues and a 3-residue C-terminal hinge <scene name='Triose_Phosphate_Isomerase/Loop6hinges/1'>Loop 6 Hinges</scene>. The complete closure of this loop, a movement of roughly 7 Å for the tip of the loop (C_α of Thr172) and occurring on a microsecond timescale, is facilitated by several hydrogen bonding interactions between loop 6 and loop 7 including H-bonds between the hydroxyl group of Tyrosine 208 (loop 7) and the amine nitrogen of Alanine 176 as well as between Serine 211 (loop 7) and Glycine 173. As mentioned above, the loop shuts when the enediol is present, effectively shielding both ligand and catalytic residues from solvent exposure, and reopens when the isomerization is complete. Site-directed mutagenesis experiments substituting a Phenylalanine for the Tyrosine resulted in a 2400-fold decrease in catalytic activity. <ref>PMID:9449311</ref> and it is beleived the opening/closing of loop 6 and loop 7 is partially rate-limiting. Additionally, extensive mechanistic and kinetic experiments involving Trypanosoma brucei, a parasitic protist causing sleeping sickness in humans, has revealed the structural and functional importance of a proline residue at position 168 in conjunction with transmitting the signal of ligand binding to the conformational change of the catalytic glutamate residue (Glu167 in ''T.brucei'') and the subsequent proper loop 6 closure.<ref>PMID:17176070</ref>  Specifically, the proline residue is positioned at the beginning of loop 6 as to aid in the catalytic glutamate side chain flipping from the inactive swung-out to the active swung-in conformation, facilitating the closure of the loop. Structurally, in the unliganded (open) conformation, the Glu-Pro peptide bond is in the energetically favored trans conformation; however, in the liganded (closed) conformation, the pyrrolidine ring of proline adopts a rare strained planar conformation (9 kJ/mol in vacuo), suggesting that the strain could be important for loop opening and product release, upon completion of the reaction cycle.<ref>PMID:12522213</ref>