Triose Phosphate Isomerase: Difference between revisions
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===Ω Loop 6=== | ===Ω Loop 6=== | ||
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/ | 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/12'>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/Loop6hinges2/3'>Loop 6 Hinges</scene>.) The complete closure of this loop, a movement of roughly 7 Å for the tip of the loop (C<sub>α</sub> 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. | ||
Site-directed mutagenesis experiments substituting a Phenylalanine for the Tyrosine resulted in a 2400-fold decrease in catalytic activity<ref>PMID:9449311</ref>. It is believed 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 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> | Site-directed mutagenesis experiments substituting a Phenylalanine for the Tyrosine resulted in a 2400-fold decrease in catalytic activity<ref>PMID:9449311</ref>. It is believed 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 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> | ||