Triose Phosphate Isomerase: Difference between revisions

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Gregg Snider, Stephen Everse, Eran Hodis, David Canner, Eric Martz, Michal Harel, Alexander Berchansky, Jane S. Richardson, Angel Herraez

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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 out tritium labeling experiments demonstrating a significant amount of intramolecular transfer (49%) of the <sup>3</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>.
-[[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.
+[[Image:LBHB2_Glu1.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.
 [[Image:crisscross2.png|right|thumb|750x350px| ''' Alternative "Criss-Cross" TPI Mechanism Involving LBHB Between Glu165 and O1 of the Intermediate''']]