| 1. |
- Hebert, Hans, et al.
(författare)
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The structure of membrane associated proteins in eicosanoid and glutathione metabolism as determined by electron crystallography
- 2007
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Ingår i: Current opinion in structural biology. - : Elsevier BV. - 0959-440X .- 1879-033X. ; 17:4, s. 396-404
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Forskningsöversikt (refereegranskat)abstract
- Membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) are involved in biosynthesis of arachidonic-derived mediators of pain, fever, and inflammation as well as in biotransformation and detoxification of electrophilic substances. Structure determination of microsomal glutathione transferase 1 using electron crystallography has provided the first atomic model of an MAPEG member. The homotrimer consists of three repeats of a four-helix transmembrane bundle with the largest extramembranous domain connecting the first and second helix and with a short proline rich loop on the same side between helices three and four. Residues of importance for intramolecular or intermolecular contacts as well as for stabilizing the active site have been identified and the results can be applied for interpreting structure-function relationship for similar MAPEG members.
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| 2. |
- Holm, Peter, et al.
(författare)
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Structural basis for detoxification and oxidative stress protection in membranes
- 2006
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 360:5, s. 934-945
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Tidskriftsartikel (refereegranskat)abstract
- Synthesis of mediators of fever, pain and inflammation as well as protection against reactive molecules and oxidative stress is a hallmark of the MAPEG superfamily (membrane associated proteins in eicosanoid and glutathione metabolism). The structure of a MAPEG member, rat mictosomal glutathione transferase 1, at 3.2 angstrom resolution, solved here in complex with glutathione by electron crystallography, defines the active site location and a cytosolic domain involved in enzyme activation. The glutathione binding site is found to be different from that of the canonical soluble glutathione transferases. The architecture of the homotrimer supports a catalytic mechanism involving subunit interactions and reveals both cytosolic and membraneous substrate entry sites, providing a rationale for the membrane location of the enzyme.
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| 3. |
- Kumar, Ramakrishnan B., et al.
(författare)
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Deciphering the Interaction of FLAP and 5LO
- 2013
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 104:2, s. 540A-540A
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 4. |
- Mittal, Monica, et al.
(författare)
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Kinetic investigation of human 5-lipoxygenase with arachidonic acid
- 2016
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Ingår i: Bioorganic & Medicinal Chemistry Letters. - : Elsevier. - 0960-894X .- 1464-3405. ; 26:15, s. 3547-3551
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Tidskriftsartikel (refereegranskat)abstract
- Human 5-lipoxygenase (5-LOX) is responsible for the formation of leukotriene (LT)A(4), a pivotal intermediate in the biosynthesis of the leukotrienes, a family of proinflammatory lipid mediators. 5-LOX has thus gained attention as a potential drug target. However, details of the kinetic mechanism of 5-LOX are still obscure. In this Letter, we investigated the kinetic isotope effect (KIE) of 5-LOX with its physiological substrate, arachidonic acid (AA). The observed KIE is 20 +/- 4 on k(cat) and 17 +/- 2 on k(cat)/K-M at 25 degrees C indicating a non-classical reaction mechanism. The observed rates show slight temperature dependence at ambient temperatures ranging from 4 to 35 degrees C. Also, we observed low Arrhenius prefactor ratio (A(H)/A(D) = 0.21) and a small change in activation energy (E-a(D) - E-a(H) = 3.6 J/mol) which suggests that 5-LOX catalysis involves tunneling as a mechanism of H-transfer. The measured KIE for 5-LOX involves a change in regioselectivity in response to deuteration at position C7, resulting in H-abstraction form C10 and formation of 8-HETE. The viscosity experiments influence the (H)k(cat), but not (D)k(cat). However the overall kcat/K-M is not affected for labeled or unlabeled AA, suggesting that either the product release or conformational rearrangement might be involved in dictating kinetics of 5-LOX at saturating conditions. Investigation of available crystal structures suggests the role of active site residues (F421, Q363 and L368) in regulating the donor-acceptor distances, thus affecting H-transfer as well as regiospecificity. In summary, our study shows that that the H-abstraction is the rate limiting step for 5-LOX and that the observed KIE of 5-LOX is masked by a change in regioselectivity.
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