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| 2. |
- Martinelle, M, et al.
(författare)
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The role of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase
- 1996
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Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139 .- 1460-213X. ; 9:6, s. 519-524
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Tidskriftsartikel (refereegranskat)abstract
- The importance of Glu87 and TRp89 in the lid of Humicola lanuginosa lipase for the hydrolytic activity at the water/lipid interface was investigated by site-directed mutagenesis. It was found that the effect on the hydrolytic activity upon the replacement of Trp89 with Phe, Leu, Gly or Glu was substrate dependent, The Trp89 mutants displayed an altered chain length specificity towards triglycerides, with a higher relative activity towards triacetin and trioctanoin compared with tributyrin, Trp89 was shown to be less important in the hydrolysis of vinyl esters compared with ethyl esters and triglycerides. An exclusive effect on the acylation reaction rate by the mutation of Trp89 was consistent with the data, It is suggested that Trp89 is important in the process of binding the acyl chain of the substrate into the active site for optimal acylation reaction rate, The Trp89Phe mutation resulted in an increased hydrolytic activity towards 2-alkylalkanoic acid esters. This is suggested to be due to reduction of unfavourable van der Waals contacts between Trp89 and the 2-substituent of the substrate, Thus, in contrast to natural substrates, Trp89 has a negative impact on the catalytic efficiency when substrates with bulky acyl chains are used, In contrast to the Trp89 mutations, the effect on the hydrolytic activity of the Glu87Ala mutation was almost substrate independent, 35-70% activity of wild-type lipase, A reduction of both the acylation and deacylation reaction was consistent with the data.
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| 3. |
- Oppermann, U, et al.
(författare)
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Regulatory factors and motifs in SDR enzymes
- 1999
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Ingår i: Advances in experimental medicine and biology. - Boston, MA : Springer US. - 0065-2598. ; 463, s. 365-371
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Tidskriftsartikel (refereegranskat)
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| 4. |
- Uppenberg, J, et al.
(författare)
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Crystallographic and molecular-modeling studies of lipase B from Candida antarctica reveal a stereospecificity pocket for secondary alcohols.
- 1995
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Ingår i: Biochemistry. - 0006-2960. ; 34:51, s. 16838-51
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Tidskriftsartikel (refereegranskat)abstract
- Many lipases are potent catalysts of stereoselective reactions and are therefore of interest for use in chemical synthesis. The crystal structures of lipases show a large variation in the shapes of their active site environments that may explain the large variation in substrate specificity of these enzymes. We have determined the three-dimensional structure of Candida antarctica lipase B (CALB) cocrystallized with the detergent Tween 80. In another crystal form, the structure of the enzyme in complex with a covalently bound phosphonate inhibitor has been determined. In both structures, the active site is exposed to the external solvent. The potential lid-forming helix alpha 5 in CALB is well-ordered in the Tween 80 structure and disordered in the inhibitor complex. The tetrahedral intermediates of two chiral substrates have been modeled on the basis of available structural and biochemical information. The results of this study provide a structural explanation for the high stereoselectivity of CALB toward many secondary alcohols.
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