Triose Phosphate Isomerase: Difference between revisions

An additional explanation of the TPI mechanism proposed by Cleeland and Kreevoy stipulates the formation of a [http://en.wikipedia.org/wiki/Low-barrier_hydrogen_bond Low-barrier hydrogen bond]<ref>PMID:8009219</ref>.  Support for this LBHB arose from the rare observation of a hydrogen bond between the carbonyl oxygen of the substrate and a ''neutral'' histidine. It was reasoned that a neutral histidine was required to match the p''K''a of the enediol, a requirement for the formation of a shorter and stronger LBHB (pKa's ~ 14). It was rationalized that this strengthened hydrogen bond and ideal geometry would effectively speed up the enolization reaction. Structural evidence for this LBHB was found in a 1.2 Å crystal structure of TIM complexed with DHAP demonstrating an extremely short hydrogen bond (2.6 Å) between His95 and O2 of DHAP <ref>PMID:12509510</ref>.  Under the mechanism stipulating a LBHB between His95 and O2 of DHAP, Glu165 would catalyze all proton transfers between C1 and C2, while His95 would act as an electrophilic catalyst by forming a close, stabilizing LBHB with the ''cis''-enediolate 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 [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>http://en.wikipedia.org/wiki/Triose_Phosphate_Isomerase_deficiency Triose Phosphate Isomerase Deficiency</ref>, 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>