Triose Phosphate Isomerase: Difference between revisions
Gregg Snider (talk | contribs) No edit summary |
Gregg Snider (talk | contribs) No edit summary |
||
| Line 14: | Line 14: | ||
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> | 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> | ||
[[Image: | [[Image:classical2.png|left|thumb|650px| '''Classic Mechanism proposed by Knowles and co-workers'''. Figure adapted from Harris ''et al''. ''Biochemistry'' 1997, 26, 14661-14675]] | ||
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 [http://en.wikipedia.org/wiki/Carboxylate 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 [http://en.wikipedia.org/wiki/Hydroxyl 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 <sup>3</sup>H 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> | 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 [http://en.wikipedia.org/wiki/Carboxylate 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 [http://en.wikipedia.org/wiki/Hydroxyl 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 <sup>3</sup>H 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> | ||
| Line 27: | Line 27: | ||
[[Image: | [[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 ]] | ||
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. | ||