Analysis of the catalyic mechanism of a fungal lipase using computer-aided design and structural mutants |
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Authors: | Beer HD; Wohlfahrt G; McCarthy JEG; Schomburg D; Schmid RD |
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Affiliation: | 1Department of Enzymology Allmandring 31, D-70569 Stuttgart,Germany
2Department of Gene Expression Allmandring 31, D-70569 Stuttgart, Germany
3Department of Molecular Structure Research, National Biotechnology Research Centre (GBF) Mascheroder Weg 1, D-38124 Braunschweig
5institut fr Technische Biochemie, Universitt Stuttgart Allmandring 31, D-70569 Stuttgart, Germany |
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Abstract: | Both an active enzyme conformation and stabilization of tetrahedraltransition states are essential for the catalysis of ester bondhydrolysis by lipases. X-ray structural data and results fromsite-directed mutagenesis experiments with proteases have beenused as a basis for predictions of amino acid residues likelyto have key functions in lipases. The gene encoding a lipasefrom Rhizopus oryzae was cloned and expressed in Escherichiacoli. Site-directed mutagenesis of this gene was used to testthe validity of computer-aided predictions of the functionalroles of specific amino acids in this enzyme. Examination ofthe kinetic constants of the Rhizopus oryzae lipase variantsallowed us to identify amino acid residues which are directlyinvolved in the catalytic reaction or which stabilize the activegeometry of the enzyme. The combination of these results withmolecular mechanics simulations, based on a homology-derivedstructural model, provided new information about structure-functionrelationships. The interpretation of the data is consistentwith results obtained with other hydrolases, such as proteases. |
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Keywords: | enzyme kinetics// homology modelling// molecular mechanics// protein variants// reaction mechanism// Rhizopus oryzae lipase// structure function relationship |
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