Triose-Phosphate Isomerase

{{STRUCTURE_2ypi| PDB=2ypi | SCENE= }}

== Overview ==

'''Triose Phosphate Isomerase''' (TPI or TIM) [EC 5.3.1.1] is a ubiquitous enzyme with a molecular weight of 56 kD which catalyzes the reversible interconversion of the triose phosphate isomers dihydroxyacetone phosphate ([http://en.wikipedia.org/wiki/DHAP 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 [http://en.wikipedia.org/wiki/Isomerization isomerization] of a ketose (DHAP) to an aldose [http://en.wikipedia.org/wiki/Glyceraldehyde_3-phosphate 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 [http://en.wikipedia.org/wiki/Glycolysis glycolysis]. TPI is an example of a [http://en.wikipedia.org/wiki/Catalytically_perfect_enzyme 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. Other catalytically perfect enzymes include [http://en.wikipedia.org/wiki/Carbonic_anhydrase carbonic anhydrase], [http://en.wikipedia.org/wiki/Acetylcholinesterase acetylcholinesterase], [http://en.wikipedia.org/wiki/Catalase catalase] and [http://en.wikipedia.org/wiki/Fumarase fumarase]. In addition to its relevance in glycolysis, TPI is also involved in metabolic biological processes such as gluconeogenesis, pentose phosphate shunt and fatty acid biosynthesis among others.

[[Image:triosejpg.jpg|right|thumb|TPI]]

== Mechanism ==

TPI catalyzes the transfer of a hydrogen atom from carbon 1 to carbon 2, an intramolecular [http://en.wikipedia.org/wiki/Oxidation_reduction oxidation-reduction] reaction. This isomerization of a ketose to an aldose proceeds through an cis-enediol intermediate. This isomerization proceeds without the need for any cofactors and the enzyme confers a 10⁹ rate enhancement relative to the nonenzymatic reaction involving carboxylate ion.<ref>PMID:2043623</ref>

=== Acid Base Catalysis ===

TPI carries out the isomerization reaction through an acid base mediated mechanism involving <scene name='Triose_Phosphate_Isomerase/Three_catalytic_residues/1'>three catalytic residues </scene>. First the PGA molecule is initially attracted to the enzyme active site by the positively charged <scene name='Triose_Phosphate_Isomerase/Lys12_shaded/1'>Lysine 12</scene>, with the resulting electrostatic interactions stabilizing the substrate. <scene name='Triose_Phosphate_Isomerase/Glu165/2'>Glutamate 165</scene> plays the role of the general base catalyst by abstracting a proton from the pro(R) position of carbon 1. However, the [http://en.wikipedia.org/wiki/Carboxylate carboxylate group]of Glutamate 165 alone does not possess the basicity to abstract a proton and requires <scene name='Triose_Phosphate_Isomerase/His95/4'>Histidine 95</scene>, the general acid, to donate a proton to C-2 to stabilize the negatively charge C-2 carbonyl group, effectively forming the endediol intermediate. At this point in the mechanism, Glutamate 165 acts as a general acid by donating its proton the C-2, while Histidine 95 now acts as a general base by abstracting a proton from the [http://en.wikipedia.org/wiki/Hydroxyl 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. Additionally, the reaction mechanism of the methylglyoxal forming enzyme [http://en.wikipedia.org/wiki/Methylglyoxal_synthase 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>

===Inhibitors of Triose Phosphate Isomerase===

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, act by labeling active site residues. There are several weak reversible inhibitors of TPI including [http://en.wikipedia.org/wiki/3-Phosphoglyceric_acid 3-Phosphoglycerate], [http://en.wikipedia.org/wiki/Glycerol_3-phosphate glycerol phosphate] and [http://en.wikipedia.org/wiki/Phosphoenolpyruvic_acid phosphoenol pyruvate], with ''K''_i values ranging from 0.2-1.3 mM.<ref>PMID:15911278</ref> Additionally, two competitive inhibitors of TPI have shown moderate effectiveness including the 'high-energy intermediate (HEI)analogue' Phosphoglycolohydroxamate (''K''_i = 6-14 μM) and the 'transition-state analogue phosphoglycolic acid (''K''_i = 3 μM).<ref>PMID:15911278</ref>

== Structure & Function ==

{{STRUCTURE_2ypi| PDB=2ypi | SCENE= Triose_Phosphate_Isomerase/Helices/1}}

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 [http://en.wikipedia.org/wiki/Beta_strand beta strands] with the N-terminus of the [http://en.wikipedia.org/wiki/Alpha_helix 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>.

[[Image:TIMBP.jpg|left|thumb|'''TIM Barrel'''. Berkovitch et al. 10.1073/pnas.0407074101.]]

===Ω Loop 6===

As mentioned earlier, TPI is 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 [http://en.wikipedia.org/wiki/Methyl_glyoxal methyl glyoxal] and [http://en.wikipedia.org/wiki/Orthophosphate 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/8'>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 loop tip spanning 5-residues and a 3-residue C-terminal hinge. The complete closure of this loop, a movement of roughly 7 Ǎ for the tip of the loop (C_α of Thr172) and occurring on a microsecond timescale, is facilitated by hydrogen bonding between the hydroxyl group of Tyrosine 208 and the amine nitrogen of Alanine 176 as well as hydrogen bonding between Serine 211 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> 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><ref>PMID:16741995</ref>

[[Image:loop6.jpg|right|thumb|700px|'''Loop 6 Bonding'''.I.Kursula ''et al'' 10.1093/protein/gzh048]]

===Entropic Effects of Ω Loop 6 Hinges===

Similar to the loop spanning residues, the Ω loop 6 hinge residues share high sequence homology amongst species. The role of both the N- and C-terminal hinge regions of Ω loop 6 have been extensively studied including the replacement of conserved hinge residues with glycine, which resulted in a 2500-fold drop in ''k''_cat. The insertion of glycine into the hinge region significantly increases the flexibility of the loop due to glycine's conformational freedom, which in turn allows the loop to sample many more conformations. This has thermodynamic ramifications as these glycine-rich hinge mutants prompted a large entropy gain (+ΔS) compared to WT, effectively altering the entropic activation energy. Specifically, WT TPI is able to overcome the initial entropic gain

(order to disorder), caused by dispelling water molecules from the active site, by forming a more ordered enzyme-substrate complex. Conversely, the glycine-rich hinge mutants again promote an initial entropy gain due to water loss but are unable to pay the entropic penalty due to their inability to bind substrate tightly. Since catalysis will only occur when the closed conformation has been sampled it reasons that the likelihood of sampling this conformation is greatly reduced with the glycine-rich hinge mutants. As its overall biological role in the enzyme, the loop hinges act to limit the motion of the loop which effectively restricts the number of conformations accessible to the enzyme. In this manner, TPI acts like an entropy trap.

== Disease ==

{{STRUCTURE_2ypi| PDB=2ypi | SCENE=Triose_Phosphate_Isomerase/Glu104/1}}

[http://en.wikipedia.org/wiki/Triose_Phosphate_Isomerase_deficiency Triose Phosphate Isomerase Deficiency], initially described in 1965, is an autosomal recessive inherited disorder with characteristics ranging from chronic haemolytic anaemia, increased susceptibility to infections, severe neurological dysfunction, and often times death in early childhood.<ref>PMID:10916682</ref> TPI has been most closely linked to a point mutation at the <scene name='Triose_Phosphate_Isomerase/Glu_104/3'>Glu104</scene> residue which results in the <scene name='Triose_Phosphate_Isomerase/Glu104asp2/1'>Glu104Asp</scene> mutation. A common marker for TPI deficiency is the increased accumulation of dihydroxyacetone phosphate in erythrocyte extracts as a result in the inability of the mutant enzyme to catalyze the isomerization to D-glyceraldehyde-3-phosphate. Recent evidence has indicated that the point mutation does not prove detrimental to the rate of catalysis of the enzyme, but rather effects the ability of the enzyme to dimerize.<ref>PMID:17183658</ref>

'''Role in Alzheimer's Disease''': Recent discoveries in Alzheimer Disease research has indicated that amyloid beta-peptide induced nitro-oxidative damage promotes the nitrotyrosination of the glycolytic enzyme triosephosphate isomerase in human neuroblastoma cells.<ref>PMID:19251756</ref> nitro-triosephosphate isomerase was found to be present in brain slides from double transgenic mice overexpressing human amyloid precursor protein as well as in Alzheimer's disease patients. Specifically, the nitrotyrosination occurs on <scene name='Triose_Phosphate_Isomerase/Two_tyrosines_shaded/2'>Tyr164 and Tyr208</scene> , which are located in close proximity to the catalytic center, and this modification correlates with a reduced isomerase activity. Additionally, according to work done by Francesc Guix and colleagues, nitro-triosphosphate isomerase contributed to the formation of large beta-sheet aggregates ''in vitro'' and ''in vivo''.

==Evolutionary Conservation==

{{STRUCTURE_2ypi| PDB=2ypi | SCENE=Triose_Phosphate_Isomerase/Conserved/1}}

[[Image:loopsequence.jpg|left|thumb|200px|'''Loop 6 & 7 Sequence Homology'''.I.Kursula ''et al'' 10.1093/protein/gzh048]]

Due to its role in the glycolysis, an essential process to many organisms, TPI has been isolated and crystallized from several species giving rise to extensive multiple alignment ''in silico'' experiments which subsequently provided <scene name='Triose_Phosphate_Isomerase/Conserved/1'>amino acid conservation structures</scene> of TPI. <ref>PMID:12403619</ref> Collectively, these tools have determined that TPI has a roughly 50% sequence conservation from bacteria to humans,<ref>PMID:8130194</ref>

== Links ==

* [[1tim]] (Triose Phosphate Isomerase from ''Gallus gallus'')

* [[2ypi]] (Complex with TPI and PGA substrate from ''Saccharomyces cerevisiae'')

* [[2vom]] (Glu104Asp mutation contributing to TPI deficiency)

* [[1wyi]] (Triose Phosphate Isomerase from ''Homo sapians'')

* [[2j27]] (Pro168Ala mutation outlining functional role of active site proline)

== References ==

<references />