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- J L Sussman, M Harel, M L Raves, K Giles, R B G Ravelli, L G M Peng, G J Kleywegt, D M Quinn, H K Nair, and I Silman
(författare)
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Studies on the 3-D structure of Torpedo Acetylcholinesterase
- 1996
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Ingår i: 1996 U.S. Army Medical Defense Bioscience Review. - : U.S. Army Medical Research Institute of Chemical Defense, Baltimore, MD. ; , s. 13-22
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Bokkapitel (refereegranskat)
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- Becker, D., et al.
(författare)
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Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum : the pH behaviour of Trichoderma reesei CeI7A and its E223S/A224H/L225V/T226A/D262G mutant
- 2001
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 356, s. 19-30
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Tidskriftsartikel (refereegranskat)abstract
- The crystal structures of Family 7 glycohydrolases suggest that a histidine residue near the acid/base catalyst could account for the higher pH optimum of the Humicola insolens endoglucanase Cel7B, than the corresponding Trichoderma reesei enzymes. Modelling studies indicated that introduction of histidine at the homologous position in T. reesei Cel7A (Ala(224)) required additional changes to accommodate the bulkier histidine side chain. X-ray crystallography of the catalytic domain of the E223S/A224H/L225V/T226A/D262G mutant reveals that major differences from the wild-type are confined to the mutations themselves, The introduced histidine residue is in plane with its counterpart in H. insolens Cel7B, but is 1.0 Angstrom (= 0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 Angstrom contact between N-2 and O'(1). The pH variation of k(cat)/K-m for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wildtype and mutant, with pK(1) shifting from 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2) shifting from 5.99+/-0.02 to 6.78+/-0.02. With this poor substrate, the ionizations probably represent those of the free enzyme. The relative k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower k(cat)/K-m values for both lactosides and cellobiosides, and a marginally lower stability. However, k(cat) values for cellobiosides are higher for the mutant. This we attribute to reduced nonproductive binding in the +1 and +2 subsites; inhibition by cellobiose is certainly relieved in the mutant. The weaker binding of cellobiose is due to the loss of two water-mediated hydrogen bonds.
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- I. Sinning, G.J. Kleywegt, S.W. Cowan, P. Reinemer, H.W. Dirr, R. Huber, G.L. Gilliland, R.N. Armstrong, X. Ji, P.G. Board, B. Olin, B. Mannervik & T.A. Jones
(författare)
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Structure determination and refinement of human alpha class glutathione transferase A1-1, and a comparison with the mu and pi class enzymes
- 1993
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Ingår i: Journal of Molecular Biology. ; 232, s. 192-212
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Tidskriftsartikel (refereegranskat)
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| 7. |
- Sinning, I, et al.
(författare)
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Structure determination and refinement of human alpha class glutathione transferase A1-1, and a comparison with the Mu and Pi class enzymes.
- 1993
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Ingår i: J Mol Biol. - 0022-2836. ; 232:1, s. 192-212
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Tidskriftsartikel (refereegranskat)abstract
- The crystal structure of human alpha class glutathione transferase A1-1 has been determined and refined to a resolution of 2.6 A. There are two copies of the dimeric enzyme in the asymmetric unit. Each monomer is built from two domains. A bound inhibitor, S-benzyl-glutathione, is primarily associated with one of these domains via a network of hydrogen bonds and salt-links. In particular, the sulphur atom of the inhibitor forms a hydrogen bond to the hydroxyl group of Tyr9 and the guanido group of Arg15. The benzyl group of the inhibitor is completely buried in a hydrophobic pocket. The structure shows an overall similarity to the mu and pi class enzymes particularly in the glutathione-binding domain". The main difference concerns the extended C terminus of the alpha class enzyme which forms an extra alpha-helix that blocks one entrance to the active site and makes up part of the substrate binding site.
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