Triose Phosphate Isomerase: Difference between revisions

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The formation and stabilization of the enediol(ate) intermediate has been a source of great discussion amongst scientists in the field. This is due to the fact that the formation of the enediol(ate) intermediate presents a large thermodynamic barrier involving the abstraction of the α-proton from the carbon acid substrate, an unfavorable process due to the lack of acidity and high p''K''a of the C1 proton of the substrate. The "classic" mechanism (shown above) put forth by Knowles and co-workers stated that the kinetic barrier is overcome by the concerted deprotonation of the C1 and the protonation of the C2 carbonyl oxygen which would effectively allow for equivalent free energies for each species shown shown, thus promoting an equilibrium constant near unity.<ref>PMID:9398185</ref> This concept of preferential binding arising from matching of the reaction energy barriers is a common theme of enzyme catalysis <ref>PMID:17287353</ref>.
The formation and stabilization of the enediol(ate) intermediate has been a source of great discussion amongst scientists in the field. This is due to the fact that the formation of the enediol(ate) intermediate presents a large thermodynamic barrier involving the abstraction of the α-proton from the carbon acid substrate, an unfavorable process due to the lack of acidity and high p''K''a of the C1 proton of the substrate. The "classic" mechanism (shown above) put forth by Knowles and co-workers stated that the kinetic barrier is overcome by the concerted deprotonation of the C1 and the protonation of the C2 carbonyl oxygen which would effectively allow for equivalent free energies for each species shown shown, thus promoting an equilibrium constant near unity.<ref>PMID:9398185</ref> This concept of preferential binding arising from matching of the reaction energy barriers is a common theme of enzyme catalysis <ref>PMID:17287353</ref>.


[[Image:TPIfreeenergy2png.png|left|thumb|300px| '''Free-Energy Profile for the Reaction Catalyzed by Triosephosphate Isomerase'''.        Figure adapted from Albery and Knowles(1976) ''Biochemistry'' 15 (25): 5627–5631.]]
[[Image:TPIfreeenergy2png.png|left|thumb|300px| '''Free-Energy Profile for the Reaction Catalyzed by Triosephosphate Isomerase'''.]]       Figure adapted from Albery and Knowles(1976) ''Biochemistry'' 15 (25): 5627–5631.]]


=== Low-Barrier Hydrogen Bond in the TPI Mechanism===
=== Low-Barrier Hydrogen Bond in the TPI Mechanism===
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[[Image:LBHB1_2.png|thumb|right|400px| '''Proposed LBHB between Histidine and Enediol Intermediate'''. Figure adapted from Cleland & Kreevoy ''Science'' Vol. 264  1994 pg.1887-1890 ]]
[[Image:LBHB1_2.png|thumb|right|400px| '''Proposed LBHB between Histidine and Enediol Intermediate'''.]]  
   
   
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.
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.


[[Image:TPImechanism2.png|thumb|right|400px| '''TPI Mechanism with LBHB between His95 and O2 of substrate'''. Figure adapted from Frey and Hegeman ''Enzymatic Reaction Mechanisms'' 2007]]
[[Image:TPImechanism2.png|thumb|right|400px| '''TPI Mechanism with LBHB between His95 and O2 of substrate'''.]]
 


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>.   
[[Image:LBHB2_Glu.png|left|thumb|500x250px|'''LBHB between Glu165 and DHAP''' Figure adapted from Harris ''et al''. ''Biochemistry'' 1997, 26, 14661-14675]] 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 the 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_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 the 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'''. Figure adapted from Harris ''et al''. ''Biochemistry'' 1997, 26, 14661-14675]]
[[Image:crisscross2.png|right|thumb|750x350px| ''' Alternative "Criss-Cross" TPI Mechanism Involving LBHB Between Glu165 and O1 of the Intermediate'''.]]


===Inhibitors of Triose Phosphate Isomerase===  
===Inhibitors of Triose Phosphate Isomerase===  
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{{STRUCTURE_2ypi|  PDB=2ypi  |  SCENE= Triose_Phosphate_Isomerase/Helices/1}}  
{{STRUCTURE_2ypi|  PDB=2ypi  |  SCENE= Triose_Phosphate_Isomerase/Helices/1}}  
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  nearly identical subunits each consisting of 247 amino acids and differing only at their N-terminal ends. Each TPI monomer 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 <scene name='Triose_Phosphate_Isomerase/Helix_shaded_sheet_3/1'>alpha helices</scene> surrounding 8 interior <scene name='Triose_Phosphate_Isomerase/Beta_sheet_labelled/1'>beta sheets</scene>, which form a conserved structural domain called a closed alpha/beta barrel (αβ) or more specifically a <scene name='Triose_Phosphate_Isomerase/Tim_barrel_2/1'>TIM Barrel</scene>, a domain estimated to be present in 10% of all enzymes. Characteristic of most all [http://en.wikipedia.org/wiki/TIM_barrel 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-terminus of the [http://en.wikipedia.org/wiki/Beta_strand beta strands] with the N-terminus of the [http://en.wikipedia.org/wiki/Alpha_helix alpha helices].TIM barrel proteins also share a structurally conserved phosphate binding motif, with the phosphate either coming from the substrate or from cofactors. <ref> http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv</ref>.
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  nearly identical subunits each consisting of 247 amino acids and differing only at their N-terminal ends. Each TPI monomer 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 <scene name='Triose_Phosphate_Isomerase/Helix_shaded_sheet_3/1'>alpha helices</scene> surrounding 8 interior <scene name='Triose_Phosphate_Isomerase/Beta_sheet_labelled/1'>beta sheets</scene>, which form a conserved structural domain called a closed alpha/beta barrel (αβ) or more specifically a <scene name='Triose_Phosphate_Isomerase/Tim_barrel_2/1'>TIM Barrel</scene>, a domain estimated to be present in 10% of all enzymes. Characteristic of most all [http://en.wikipedia.org/wiki/TIM_barrel 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-terminus of the [http://en.wikipedia.org/wiki/Beta_strand beta strands] with the N-terminus of the [http://en.wikipedia.org/wiki/Alpha_helix alpha helices].TIM barrel proteins also share a structurally conserved phosphate binding motif, with the phosphate either coming from the substrate or from cofactors. <ref> http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv</ref>.
[[Image:beta barrel.png|left|thumb|'''TIM Barrel'''.Figure adapted from Berkovitch et al. PNAS 101 (45) 15870-15875, 2004.]]
[[Image:beta barrel.png|left|thumb|'''TIM Barrel'''.]]


===Ω Loop 6===
===Ω Loop 6===

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