Triose Phosphate Isomerase: Difference between revisions

TPI catalyzes the transfer of a hydrogen atom from carbon 1 to carbon 2, an intramolecular [http://en.wikipedia.org/wiki/Oxidation_reduction oxidation-reduction] reaction. This isomerization of a ketose to an aldose proceeds through an ''cis''-enediol(ate) intermediate. This isomerization proceeds without any cofactors and the enzyme confers a 10⁹ rate enhancement relative to the nonenzymatic reaction involving a chemical base (acetate ion).<ref>PMID:2043623</ref>

=== Acid-Base Catalysis ===

The mechanism of TPI has been extensively studied by prominent enzymologists for several decades leading to several different proposed mechanisms of catalysis, a few of which will be discussed in the following section. The original "Classic" mechanism put forth by Knowles and co-workers is outlined in the mechanism provided below.<ref>PMID:9398185</ref>   

{{STRUCTURE_2ypi|PDB=2ypi|SCENE=Triose_Phosphate_Isomerase/Three_catalytic_residues/3}}  

TPI carries out the isomerization reaction through an acid-base-mediated mechanism involving <scene name='Triose_Phosphate_Isomerase/Three_catalytic_residues/3'>three catalytic residues</scene>.  First the DHAP or GAP subtrate is initially attracted to the enzyme active site through electrostatic interactions between the negatively charged substrate phosphate group and the positively charged <scene name='Triose_Phosphate_Isomerase/Lys12_shaded/1'>Lys12</scene>, with the resulting interaction stabilizing the substrate. According to the "classic" mechanism, <scene name='Triose_Phosphate_Isomerase/Glu165/3'>Glu165</scene> plays the role of the general base catalyst by abstracting a proton from the pro(''R'') position of carbon 1 of DHAP or the C-2 proton of GAP. However, the carboxylate group of Glutamate 165 alone does not possess the basicity to abstract a proton and requires <scene name='Triose_Phosphate_Isomerase/His95/6'>His95</scene>, the general acid, to donate a proton to stabilize the negative charge building up on C-2 carbonyl oxygen, effectively stabilizing the planar endediol(ate) intermediate,.  Lys12 and Asn11 also function to stabilize the negative charge which builds up on this intermediate. At this point in the mechanism, Glutamate 165 acts as a general acid by donating its proton to the neighboring C-2, while Histidine 95 now acts as a general base by abstracting a proton from the hydroxyl group of C-1. The final step in the reaction is the formation of the GAP isomer product while glutamate and histidine are returned to their original forms, regenerating the enzyme.  In studies using tritium labeled DHAP, Knowles observed only ~ 6% intramolecular transfer of the ³H label to the GAP product.  In explaining this result, Knowles argued that the hydrogen bound to the Glu165 was in equilibrium with those in bulk solvent.  Additionally, the reaction mechanism of the methylglyoxal forming enzyme [http://en.wikipedia.org/wiki/Methylglyoxal_synthase methylglyoxal synthase (MGS)] is believed to be similar to that of triosephosphate isomerase. Both enzymes utilize DHAP to form an enediol(ate) phosphate intermediate as the first step of their reaction pathways; however, the second catalytic step in the MGS reaction pathway features the elimination of phosphate and collapse of the enediol(ate) to form methylglyoxal rather then reprotonation to form the isomer glyceraldehyde 3-phosphate as seen in TPI.<ref>PMID:10368300</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>.

=== Low-Barrier Hydrogen Bond in the TPI Mechanism===

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.

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 ¹H 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>.   

===Inhibitors of Triose Phosphate Isomerase===  

{{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>.

===Ω Loop 6===