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Chiral recognition on cellulases: The active sites of the enzymes are involved
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry.
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Two cellobiohydrolases (CBH I and CBH II) and endoglucanase I (EG I) fromTrichoderma reesei, and CBH 58 from Phanerochaete chrysosporium were immobilized onsilica as chiral stationary phases (CSPs) in HPLC for enantioseparation of a group ofpharmaceutical compounds. CBH I and CBH 58 wore excellent chiral selectors for β-adrenergic blocking agents, whereas CBH II displayed good enantioselectivity for someother compounds The CBHs share the common feature that their active sites are located intunnels. EG I, with an open groove-shaped active site, had much poorer enantioselectivitythan CBH I, even though these proteins are highly homologous, indicating that a tunnel-shaped active site is important for the chiral recognition ability of the cellulase.

The chiral compounds separated on the cellulase-CSPs can influence the catalyticactivity of the cellulases as either inhibitors (in most cases) or activators (some cases inCBH II). The β-blockers were competitive inhibitors of CBH I, EG I and CBH 58, and theirinhibition constants showed that the stronger inhibitors were also the enantiomers that weremore retained on the cellulase-CSPs. The consistency of the enzyme kinetics with thechromatography revealed that the active sites of the cellulases coincided with the chiralrecognition sites. The impairment of the enantioselectivities of the catalytically deficientmutants of CBH I (E212Q, D214N and E217Q) paralleled the loss of their enzymaticactivities, suggesting that the catalytic carboxylate residues (Glu212, Asp214 and Glu217)were directly involved in the binding and the chiral discrimination. Amidation of all thecarboxylate groups of CBH I-CSP abolished both the enantioselectivity and the enzymaticactivity of the CSP, but these properties could be protected from the modification by thepresence of cellobiose that binds in the active site. The crystal structure of the catalyticdomain of CBH I with bound (S)-propranolol revealed, for the first time, the bindingbetween an enantiomer and a protein used as chiral selector at a molecular level.

Using cellobiose or lactose as selective competitors in chromatography, the totalbinding sites of the enantiomers on the cellulase-CSPs could be resolved into two classes,namely enantioselective sites that were blocked by the competitor and non-selective sitesthat were unaffected. Their relationship is well described by a mathematical model.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2000. , 53 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 506
Keyword [en]
Keyword [sv]
National Category
Biochemistry and Molecular Biology
Research subject
URN: urn:nbn:se:uu:diva-1027ISBN: 91-554-4643-4OAI: oai:DiVA.org:uu-1027DiVA: diva2:160563
Public defence
2000-02-18, Lecture room B42, Biomedical Center (BMC), Uppsala, Uppsala, 13:30
Available from: 2000-01-28 Created: 2000-01-28Bibliographically approved

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