Triosephosphate isomerase
Triosephosphate isomerase (TIM) is one of the most extensively characterised enzymes in the chemical literature. TIM is a glycolytic enzyme that catalyses the central reaction in the glycolytic pathway, the interconversion of D-glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) with exceptionally high efficiency, while suppressing elimination of orthophosphate. It contains a large-scale catalytic loop motion, which alternates in open and closed positions. It is a homodimer and only the homodimer is fully active. TIM is known to exist in an unliganded and liganded conformation and the loop-6, in particular, would undergo the major conformational changes when there is a substrate or inhibitor bound at the active site. In addition, the shift of loop-6 preferentially stabilises the enediole phosphate intermediate.
Its deficiency has been shown to enhance levels of dihydroxyacetone phosphate in humans and cause chronic anaemia and neuromuscular impairment. Due to its important place in glycolysis, TIM also forms an attractive target for drug design against parasites that have the ability to survive in the mammalian bloodstream. Among the various diseases currently ravaging the tropical world, those due to such parasites are some of the most serious ones (e.g., malaria and sleeping sickness). Control of these diseases is presently a major problem and has been a focus of attention of various agencies.
Reference Protein and Structure
- Sequence
-
P00940
(4.2.3.3, 5.3.1.1)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Gallus gallus (Chicken)
- PDB
-
1tph
- 1.8 ANGSTROMS CRYSTAL STRUCTURE OF WILD TYPE CHICKEN TRIOSEPHOSPHATE ISOMERASE-PHOSPHOGLYCOLOHYDROXAMATE COMPLEX
(1.8 Å)
- Catalytic CATH Domains
-
3.20.20.70
(see all for 1tph)
Enzyme Reaction (EC:5.3.1.1)
Enzyme Mechanism
Introduction
To accomplish this isomerization, TIM first extracts a pro-R hydrogen from the C1 of DHAP and then stereospecifically introduces a proton onto the C2 atom. The flexible loop formed by residues 167-177 allows the active site to organise around substrate, and it's distortion has been shown to reduce catalytic activity and affinity for the reaction intermediate.
Glu 165 acts as a general base to abstract the pro-R proton from C1 of DHAP, with electrophilic catalysis by His 95 and possibly also Asn 11 and Lys 13 serving to make the hydrogen more acidic and to stabilise the partial negative charge that forms on the carbonyl oxygen in the transition state. Formation of the cis-enediol intermediate is aided by His 95 also acting as a general acid to donate a proton to the substrate carbonyl oxygen. Then Glu 165 transfers a proton back to C2 of the intermediate to give GAP [PMID:19348462][PMID:9843453].
Catalytic Residues Roles
| UniProt | PDB* (1tph) | ||
| Glu165 | Glu165(164)1(A) | Acts as a general base in the initial deprotonation of C1 of DHAP, then reprotonates C2 in the final step to produce GAP. It also forms the enediol intermediate by transfer of a proton to O2 and abstraction of a proton from O1 | activator, proton acceptor, proton donor |
| Asn11 | Asn11(10)1(A) | Plays an electrostatic stabilising role in the first proton abstraction and the last proton transfer step to convert EDT2 to GAP. | electrostatic stabiliser |
| Gly171 (main-N), Ser211 (main-N) | Gly171(170)1(A) (main-N), Ser211(210)1(A) (main-N) | Act to stabilise the transition state through hydrogen bonding. These residues are part of the loop and are also involved in stabilising the closed conformation of the protein. | electrostatic stabiliser |
| Glu97 | Glu97(96)1(A) | Exact role in the proposal unclear. It is likely involved in holding Lys13 and His95 in their correct orientations such that the reaction can occur. | steric role |
| Lys13 | Lys13(12)1(A) | Forms a stabilising interaction between its protonated (cationic) side chain and the negative charge that develops at the enolate-like oxygen (O2) in the transition state for deprotonation of the sugar substrate. Acts to stabilise the EDT1 intermediate. | attractive charge-charge interaction, hydrogen bond donor, electrostatic stabiliser |
| His95 | His95(94)1(A) | The hydrogen bond between His 95 and O2 in the carbonyl bond introduces a favourable polarising effect in the conversion from DHAP to EDT1. | hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, activator |
Chemical Components
proton transfer, assisted keto-enol tautomerisation, native state of enzyme regeneratedReferences
- Richard JP (2012), Biochemistry, 51, 2652-2661. A Paradigm for Enzyme-Catalyzed Proton Transfer at Carbon: Triosephosphate Isomerase. DOI:10.1021/bi300195b. PMID:22409228.
- Zhai X et al. (2015), J Am Chem Soc, 137, 15185-15197. Role of Loop-Clamping Side Chains in Catalysis by Triosephosphate Isomerase. DOI:10.1021/jacs.5b09328. PMID:26570983.
- Zhai X et al. (2014), J Phys Org Chem, 27, 269-276. Mechanistic imperatives for deprotonation of carbon catalyzed by triosephosphate isomerase: enzyme activation by phosphite dianion,. DOI:10.1002/poc.3195. PMID:24729658.
- Zhai X et al. (2013), Biochemistry, 52, 5928-5940. Structural Mutations That Probe the Interactions between the Catalytic and Dianion Activation Sites of Triosephosphate Isomerase. DOI:10.1021/bi401019h. PMID:23909928.
- Samanta M et al. (2011), Chembiochem, 12, 1886-1896. Revisiting the Mechanism of the Triosephosphate Isomerase Reaction: The Role of the Fully Conserved Glutamic Acid 97 Residue. DOI:10.1002/cbic.201100116. PMID:21671330.
- Go MK et al. (2010), Biochemistry, 49, 5377-5389. Role of Lys-12 in Catalysis by Triosephosphate Isomerase: A Two-Part Substrate Approach. DOI:10.1021/bi100538b. PMID:20481463.
- Wang Y et al. (2009), Biochemistry, 48, 4548-4556. Role of Loop−Loop Interactions in Coordinating Motions and Enzymatic Function in Triosephosphate Isomerase. DOI:10.1021/bi9002887. PMID:19348462.
- Guallar V et al. (2004), J Mol Biol, 337, 227-239. Computational Modeling of the Catalytic Reaction in Triosephosphate Isomerase. DOI:10.1016/j.jmb.2003.11.016. PMID:15001364.
- Cui Q et al. (2003), Adv Protein Chem, 66, 315-372. Catalysis and Specificity in Enzymes: A Study of Triosephosphate Isomerase and Comparison with Methyl Glyoxal Synthase. DOI:10.1016/s0065-3233(03)66008-0. PMID:14631822.
- Aparicio R et al. (2003), J Mol Biol, 334, 1023-1041. Closed conformation of the active site loop of rabbit muscle triosephosphate isomerase in the absence of substrate: evidence of conformational heterogeneity. PMID:14643664.
- Harris TK et al. (1998), Biochemistry, 37, 16828-16838. Proton Transfer in the Mechanism of Triosephosphate Isomerase†. DOI:10.1021/bi982089f. PMID:9843453.
- Zhang Z et al. (1994), Biochemistry, 33, 2830-2837. Crystal Structure of Recombinant Chicken Triosephosphate Isomerase-Phosphoglycolohydroxamate Complex at 1.8-.ANG. Resolution. DOI:10.1021/bi00176a012. PMID:8130195.
- Wierenga RK et al. (1992), J Mol Biol, 224, 1115-1126. Comparison of the refined crystal structures of liganded and unliganded chicken, yeast and trypanosomal triosephosphate isomerase. DOI:10.1016/0022-2836(92)90473-w. PMID:1569570.
- Knowles JR (1991), Nature, 350, 121-124. Enzyme catalysis: not different, just better. DOI:10.1038/350121a0. PMID:2005961.
- Pompliano DL et al. (1990), Biochemistry, 29, 3186-3194. Stabilization of a reaction intermediate as a catalytic device: definition of the functional role of the flexible loop in triosephosphate isomerase. DOI:10.1021/bi00465a005. PMID:2185832.
- Blacklow SC et al. (1990), Biochemistry, 29, 4099-4108. How can a catalytic lesion be offset? The energetics of two pseudorevertant triosephosphate isomerases. PMID:2361134.
- Lolis E et al. (1990), Biochemistry, 29, 6609-6618. Structure of yeast triosephosphate isomerase at 1.9-.ANG. resolution. DOI:10.1021/bi00480a009. PMID:2204417.
- Lolis E et al. (1990), Biochemistry, 29, 6619-6625. Crystallographic analysis of the complex between triosephosphate isomerase and 2-phosphoglycolate at 2.5-.ANG. resolution: implications for catalysis. DOI:10.1021/bi00480a010. PMID:2204418.
Step 1. Glu165 acts as the catalytic base, abstracting a proton from the alpha-carbonyl carbon.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Ser211(210)1(A) (main-N) | electrostatic stabiliser |
| Gly171(170)1(A) (main-N) | electrostatic stabiliser |
| Asn11(10)1(A) | electrostatic stabiliser |
| Glu97(96)1(A) | steric role |
| Lys13(12)1(A) | electrostatic stabiliser |
| Glu165(164)1(A) | proton acceptor |
Chemical Components
proton transfer, assisted keto-enol tautomerisation
Step 2. The enolate deprotonates His95. The deprotonation of neutral His95 creates a more favourable hydrogen bond between the residue and the enol-transition state than if the residue were protonated [PMID:9843453].
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Asn11(10)1(A) | electrostatic stabiliser |
| Gly171(170)1(A) (main-N) | electrostatic stabiliser |
| Glu97(96)1(A) | steric role |
| His95(94)1(A) | activator, hydrogen bond donor |
| Glu165(164)1(A) | activator |
| Ser211(210)1(A) (main-N) | hydrogen bond donor, electrostatic stabiliser |
| Lys13(12)1(A) | attractive charge-charge interaction, electrostatic stabiliser |
| His95(94)1(A) | proton donor |
Chemical Components
proton transfer
Step 3. His95 now acts as the catalytic base towards the enol, forming an aldehyde with concomitant reprotonation of C2 in a stereoselective manner.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Glu97(96)1(A) | steric role |
| His95(94)1(A) | activator, hydrogen bond acceptor |
| Glu165(164)1(A) | activator |
| Lys13(12)1(A) | electrostatic stabiliser, hydrogen bond donor |
| Ser211(210)1(A) (main-N) | hydrogen bond donor, electrostatic stabiliser |
| Glu165(164)1(A) | proton donor |
| His95(94)1(A) | proton acceptor |
Chemical Components
assisted keto-enol tautomerisation, proton transfer, native state of enzyme regenerated
Download: Introduction
In this proposal, Lys13 is the electrophile, rather than His95. In this mechanism, the first step is the same (Glu165 acts as the catalytic base). Then Lys13 acts as the electrophile, through the conserved Glu97, to reprotaonate the enolate. This cases His95 to rotate in the active site, correctly positioning it to abstract the proton from the terminal hydroxyl group, resulting in the re-protonation of C2 by Glu165.
Catalytic Residues Roles
| UniProt | PDB* (1tph) | ||
| Glu165 | Glu165(164)1(A) | Acts as a general base in the initial deprotonation of C1 of DHAP, then reprotonates C2 in the final step to produce GAP. It also forms the enediol intermediate by transfer of a proton to O2 and abstraction of a proton from O1 | proton acceptor, proton donor |
| Asn11 | Asn11(10)1(A) | Plays an electrostatic stabilising role in the first proton abstraction and the last proton transfer step to convert EDT2 to GAP. | electrostatic stabiliser |
| Gly171 (main-N), Ser211 (main-N) | Gly171(170)1(A) (main-N), Ser211(210)1(A) (main-N) | Act to stabilise the transition state through hydrogen bonding. These residues are part of the loop and are also involved in stabilising the closed conformation of the protein. | electrostatic stabiliser |
| Glu97 | Glu97(96)1(A) | Acts as a general acid/base, activating Lys13 to act as the electrophile in the second step of the reaction. | proton acceptor, proton donor |
| Lys13 | Lys13(12)1(A) | Acts as the electrophile and proton relay with Glu97. Also acts to stabilise the reactive intermediates. | proton relay, proton acceptor, proton donor |
| His95 | His95(94)1(A) | Acts as a general acid/base in the final deprotonation of the reaction. | proton acceptor, proton donor |
Chemical Components
assisted keto-enol tautomerisation, proton transfer, proton relay, native state of enzyme regenerated, inferred reaction stepReferences
- Samanta M et al. (2011), Chembiochem, 12, 1886-1896. Revisiting the Mechanism of the Triosephosphate Isomerase Reaction: The Role of the Fully Conserved Glutamic Acid 97 Residue. DOI:10.1002/cbic.201100116. PMID:21671330.
- Cui Q et al. (2003), Adv Protein Chem, 66, 315-372. Catalysis and Specificity in Enzymes: A Study of Triosephosphate Isomerase and Comparison with Methyl Glyoxal Synthase. DOI:10.1016/s0065-3233(03)66008-0. PMID:14631822.
Step 1. Glu165 acts as the catalytic base, abstracting a proton from the alpha-carbonyl carbon.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Asn11(10)1(A) | electrostatic stabiliser |
| Gly171(170)1(A) (main-N) | electrostatic stabiliser |
| Ser211(210)1(A) (main-N) | electrostatic stabiliser |
| Glu165(164)1(A) | proton acceptor |
Chemical Components
assisted keto-enol tautomerisation, proton transfer
Step 2. The enolate ion deprotonates Lys13, with concomitant deprotonation of Glu97. This causes His95 to rotate.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Lys13(12)1(A) | proton relay |
| Asn11(10)1(A) | electrostatic stabiliser |
| Gly171(170)1(A) (main-N) | electrostatic stabiliser |
| Ser211(210)1(A) (main-N) | electrostatic stabiliser |
| Lys13(12)1(A) | proton donor, proton acceptor |
| Glu97(96)1(A) | proton donor |
Chemical Components
proton transfer, proton relay
Step 3. His95 now deprotonates the terminal hydroxyl group in an assisted tautomerisation with concomitant deprotonation of Glu165.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Asn11(10)1(A) | electrostatic stabiliser |
| Gly171(170)1(A) (main-N) | electrostatic stabiliser |
| Ser211(210)1(A) (main-N) | electrostatic stabiliser |
| His95(94)1(A) | proton acceptor |
| Glu165(164)1(A) | proton donor |
Chemical Components
proton transfer, assisted keto-enol tautomerisation
Catalytic Residues Roles
| Residue | Roles |
|---|---|
| Glu97(96)1(A) | proton acceptor |
| His95(94)1(A) | proton donor |
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