Triose Phosphate Isomerase: Difference between revisions
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An additional explanation of the TPI mechanism involving the formation of a [http://en.wikipedia.org/wiki/Low-barrier_hydrogen_bond Low-barrier hydrogen bond] has been proposed by Cleeland and Kreevoy<ref>PMID:8009219</ref>. The 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, Glu2 165 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. | An additional explanation of the TPI mechanism involving the formation of a [http://en.wikipedia.org/wiki/Low-barrier_hydrogen_bond Low-barrier hydrogen bond] has been proposed by Cleeland and Kreevoy<ref>PMID:8009219</ref>. The 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, Glu2 165 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. | ||
[[Image:mechanism4.png|thumb|left|400px| '''TPI Mechanism with LBHB between His95 and O2 of substrate''' Frey and Hegeman ''Enzymatic Reaction | [[Image:mechanism4.png|thumb|left|400px| '''TPI Mechanism with LBHB between His95 and O2 of substrate''' Frey and Hegeman ''Enzymatic Reaction Mechanisms'' 2007]] | ||
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More recently a series of NMR experiments carried out by Mildvan and co-workers have shed light onto an alternative "Criss-cross" mechanism involving a LBHB between the catalytic Glu165 and the O1 oxygen of the substrate. This mechanism stipulates the His95 side chain does not directly transfer protons, this rather being accomplished entirely by Glu165. Support for this mechanism was provided by Richard and coworkers who carried tritium labeling experiments demonstrating a significant amount of intramolecular transfer (49%) of the <sup>1</sup>H label from substrate (DHAP) to product (GAP)<ref>PMID: 15709774</ref>. Using phosphoglycolohydroxamate (PGH), a mimic of the enediol(ate) intermediate, a 14.9 ppm chemical shift (6 ppm downfield) as well as a deuterium fractionation factor of 0.38 was observed with the TIM-PGH complex, corresponding to a highly deshielded proton involved in a LBHB between Glu165 and the hydroxamate oxygen of PGH. Conversely, the same NMR study found an additional hydrogen bond between the N-ε proton of His95 and the carbonyl oxygen of PGH; however, its chemical shift of 13.5 (0.4 ppm downfield from free enzyme) and fractionation factor of 0.71 indicated this was a strong H-bond, but not a LBHB.<ref>PMID:9748211</ref>. | More recently a series of NMR experiments carried out by Mildvan and co-workers have shed light onto an alternative "Criss-cross" mechanism involving a LBHB between the catalytic Glu165 and the O1 oxygen of the substrate. This mechanism stipulates the His95 side chain does not directly transfer protons, this rather being accomplished entirely by Glu165. Support for this mechanism was provided by Richard and coworkers who carried tritium labeling experiments demonstrating a significant amount of intramolecular transfer (49%) of the <sup>1</sup>H label from substrate (DHAP) to product (GAP)<ref>PMID: 15709774</ref>. Using phosphoglycolohydroxamate (PGH), a mimic of the enediol(ate) intermediate, a 14.9 ppm chemical shift (6 ppm downfield) as well as a deuterium fractionation factor of 0.38 was observed with the TIM-PGH complex, corresponding to a highly deshielded proton involved in a LBHB between Glu165 and the hydroxamate oxygen of PGH. Conversely, the same NMR study found an additional hydrogen bond between the N-ε proton of His95 and the carbonyl oxygen of PGH; however, its chemical shift of 13.5 (0.4 ppm downfield from free enzyme) and fractionation factor of 0.71 indicated this was a strong H-bond, but not a LBHB.<ref>PMID:9748211</ref>. | ||