Triose Phosphate Isomerase: Difference between revisions

<StructureSection load='2ypi' size='450' frame='true' align='right'

caption='TPI (yeast) at 2.5 &Aring; [[resolution]] ([[2ypi]]) dimer. The two identical chains are different colors. Ligand is the inhibitor 2-phosphoglycolic acid (PGA). '

[[Triose Phosphate Isomerase]] (TPI or TIM) is a ubiquitous dimeric enzyme with a molecular weight of ~54 kD (27 kD per subunit) which catalyzes the reversible interconversion of the triose phosphate isomers <scene name='Triose_Phosphate_Isomerase/Morph/1'>dihydroxyacetone phosphate (DHAP)</scene> and <scene name='Triose_Phosphate_Isomerase/Morph/2'>D-glyceraldehyde-3-phosphate (GAP)</scene>, an essential process in the glycolytic pathway. More simply, the enzyme catalyzes the <scene name='Triose_Phosphate_Isomerase/Morph/3'>isomerization of a ketose (DHAP) to an aldose (GAP)</scene>, 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.  The TPI tertiary structure is the classic example of the "TIM barrel" fold (see image at left).  The TPI structure is shown on the right (PDB entry [[2ypi]]) in complex with the inhibitor 2-phosphoglycolic acid (PGA), which is bound to each of its two active sites. 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 limited only by the substrate diffusion rate.  

In addition to its role in glycolysis, TPI is also involved in several additional metabolic biological processes including gluconeogenesis, the pentose phosphate shunt, and fatty acid biosynthesis.  A point mutation to a glutamate residue (Glu104) of TPI results in triose phosphate isomerase deficiency, an autosomal recessive inherited disorder characterized by an increased accumulation of DHAP in erythrocytes. Structurally, this point mutation abolishes TPI’s ability to dimerize, subsequently inhibiting its catalytic activity.  More details in [[ Triose Phosphate Isomerase Structure & Mechanism]] and [[Triosephosphate Isomerase]].

TPI carries out the isomerization reaction through an acid-base-mediated mechanism involving (<scene name='Triose_Phosphate_Isomerase/Three_catalytic_residues1/3'>three catalytic residues</scene>), each of which <scene name='Triose_Phosphate_Isomerase/Three_catalytic_residues2/2'>contacts the substrate</scene>.  First, the DHAP or GAP substrate is initially attracted to the enzyme active site through '''electrostatic interactions''' between the negatively charged phosphate group of the substrate and the positively charged '''Lys12''', with the resulting interaction stabilizing the substrate.

[[Image:LBHB2_Glu.png|left|thumb|500x250px|'''LBHB between Glu165 and DHAP''']] The formation of the LBHB between Glu165 and O1 of the inhibitor PGH is due to the matching of p''K''as and the alternative mechanism suggests that Glu-165, in addition to its role in initially abstracting the proton from the substrate, may also shuttle protons to and from the oxygens in the intermediate.  Also, the "criss-cross" mechanism implies that, by donating a normal hydrogen bond, the role of His95 is to polarize the carbonyl oxygen and lower the p''K''a of PGH in order to facilitate subsequent proton abstraction<ref>PMID:9748211</ref>. It has been argued that that the LBHB formed between Glu165 and PGH is a consequence of using the inhibitor PGH, whose hydroxamate p''K''a of 9 better matches Glu165 then His95, and that the biological reaction would instead see the enediol forming a LHBH with His95, as mentioned above. Overall, the mechanism employed by TPI has yet to be completely solved and recent NMR studies involving both WT and mutant TPI enzymes have revealed contributions from both the "classic" and "criss-cross" mechanisms.  

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 sequence-identical subunits (chains) each comprising 247 amino acids. Each TPI monomer (chain) 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 <font color="#ff0080">'''alpha helices'''</font> surrounding 8 interior <font color="#d0a000">'''beta strands'''</font> (<scene name='Triose_Phosphate_Isomerase/Helix_shaded_sheet_6/3'>restore initial scene</scene>), which form a conserved structural domain called a closed alpha/beta barrel (αβ) or more specifically a '''TIM barrel'''. The TIM barrel was originally named after TPI and is estimated to be present in 10% of all enzymes. Characteristic of most all 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-termini of the beta strands with the N-termini of the alpha helices. TIM barrel proteins also share a structurally conserved phosphate binding motif, with the phosphate group found in the substrate or cofactors. <ref> http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv</ref>.

In each chain, nonpolar amino acids pointing inward from the beta strands contribute to the hydrophobic core of the structure (<scene name='Triose_Phosphate_Isomerase/Hydrophobicity_plus_cartoon/2'>hydrophobic/polar distribution of beta strands</scene>). The alpha helices are amphipathic: their outer (water-contacting) surfaces are polar, while their inner surfaces are largely hydrophobic (<scene name='Triose_Phosphate_Isomerase/Hydrophobicity_plus_cartoon/4'>hydrophobic/polar distribution of alpha helices</scene>). The loops are a mixture (<scene name='Triose_Phosphate_Isomerase/Hydrophobicity_plus_cartoon/6'>hydrophobic/polar distribution of loops</scene>).

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 (omega loop) referred to as <scene name='Triose_Phosphate_Isomerase/Morph_tpi/10'>Loop 6</scene>. Loop 6 spans residues 168-178<ref>PMID:2402636</ref>, (numbering varies with species) and 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/Loop6hinges2/2'>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 bonds|hydrogen bonding]] interactions between loop 6 and loop 7. These include 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.

'''Triose Phosphate Isomerase Deficiency''': Initially described in 1965,  TPI deficiency 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> The effects of TPI deficiency are most closely linked to a point mutation at the <scene name='Triose_Phosphate_Isomerase/Glu_104_1/7'>Glu104</scene> residue which results in the <scene name='Triose_Phosphate_Isomerase/Glu104asp3/2'>Glu104Asp</scene> mutation. A common marker for TPI deficiency is the increased accumulation of DHAP in erythrocyte extracts as a result in the inability of the mutant enzyme to catalyze the isomerization to GAP. 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 have indicated that amyloid beta-peptide induced nitro-oxidative damage promotes the nitrotyrosination of TPI in human neuroblastoma cells.<ref>PMID:19251756</ref> Nitrosylated TPI was found to be present in brain slides from double transgenic mice over-expressing human amyloid precursor protein as well as in Alzheimer's disease patients. Specifically, the nitrotyrosination occurs on <scene name='Triose_Phosphate_Isomerase/Two_tyrosines_shaded1/1'>Tyr164 and Tyr208</scene> , which are located in close proximity to the catalytic center, and this modification correlates with reduced isomerization activity. Additionally, Francesc Guix and colleagues have shown nitrosylated TPI contributed to the formation of large beta-sheet aggregates ''in vitro'' and ''in vivo''.

TPI has a roughly 50% sequence conservation from bacteria to humans.<ref>PMID:8130194</ref> The <scene name='Triose_Phosphate_Isomerase/Conserved1/1'>3D pattern of amino acid conservation</scene> ([[2ypi]]) shows dramatic conservation around the catalytic site. Glu104 is also highly conserved, as are several residues in the [[#Why is the enzyme an obligate dimer?|interdigitating loop]]. Curiously, two Arg residues on the surface, distant from the dimer contact and the catalytic side, are also highly conserved. (See note about the conservation calculation<ref>The conservation pattern shown was calculated by [[ConSurfDB_vs._ConSurf|ConSurfDB]] and might obscure some conservation due to [[Evolutionary_Conservation#ConSurf-DB_Often_Obscures_Some_Functional_Sites|inclusion of proteins of different functions]]. However in the case of [[2ypi]], all sequences used in the multiple sequence alignment were TPI sequences. A manual run at the ConSurf Server, using 500 TPI sequences, gave a nearly identical result. Both runs gave an average pairwise distance close to 1.0. Hence, the conservation pattern shown is correct for TPI.</ref>.)