Triose Phosphate Isomerase: Difference between revisions

[[Triose Phosphate Isomerase]] (TPI or TIM) [5.3.1.1] is a ubiquitous enzyme with a molecular weight of roughly 54 kD (27 kD per subunit) which catalyzes the reversible interconversion of the triose phosphate isomers dihydroxyacetone phosphate DHAP and D-glyceraldehyde-3-phosphate <scene name='Triose_Phosphate_Isomerase/Pga/1'>(GAP)</scene>, an essential process in the glycolytic pathway. More simply, the enzyme catalyzes the isomerization of a ketose (DHAP) to an aldose GAP also referred to as PGAL. In regards to the two isomers, at equilibrium, roughly 96% of the triose phosphate is in the DHAP isomer form; however, the isomerization reaction proceeds due to the rapid removal of GAP from the subsequent reactions of glycolysis. TPI is an example of a catalytically perfect enzyme], indicating that for almost every enzyme-substrate encounter, a product is formed and that this interaction is only limited by the substrate diffusion rate. Diffusion as the rate-limiting step was experimentally confirmed through the use of viscogens such as glycerol and sucrose. Other catalytically perfect enzymes include carbonic anhydrase, acetylcholinesterase, catalase, and fumarase. In addition to its relevance in glycolysis, TPI is also involved in several additional metabolic biological processes including gluconeogenesis, the pentose phosphate shunt, and fatty acid biosynthesis.

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>

Triose Phosphate Isomerase is a member of the all alpha and beta (α/β) class of proteins and it is a homodimer consisting of two  nearly identical subunits each consisting of 247 amino acids and differing only at their N-terminal ends. Each TPI monomer contains the full set of catalytic residues; however, the enzyme is only active in the oligomeric form. <ref>PMID:18562316</ref> Therefore, dimerization is essential for full function of the enzyme even though it is not believed that any cooperativity exists between the two active sites.<ref>PMID: 2065677</ref> Each subunit contains 8 exterior <scene name='Triose_Phosphate_Isomerase/Helix_shaded_sheet_3/1'>alpha helices</scene> surrounding 8 interior <scene name='Triose_Phosphate_Isomerase/Beta_sheet_labelled/1'>beta sheets</scene>, which form a conserved structural domain called a closed alpha/beta barrel (αβ) or more specifically a <scene name='Triose_Phosphate_Isomerase/Tim_barrel_2/1'>TIM Barrel</scene>, a domain estimated to be present in 10% of all enzymes. Characteristic of most all [http://en.wikipedia.org/wiki/TIM_barrel TIM barrel] domains is the presence of the enzyme's active site in the lower loop regions created by the eight loops that connect the C-terminus of the beta strands with the N-terminus of the alpha helices. TIM barrel proteins also share a structurally conserved phosphate binding motif, with the phosphate either coming from the substrate or from cofactors. <ref> http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv</ref>.

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 [http://en.wikipedia.org/wiki/Trypanosoma_brucei 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>