Analysis of the catalytic mechanism of a fungal lipase using computer-aided design and structural mutants.
Both an active enzyme conformation and stabilization of tetrahedral transition states are essential for the catalysis of ester bond hydrolysis by lipases. X-ray structural data and results from site-directed mutagenesis experiments with proteases have been used as a basis for predictions of amino acid residues likely to have key functions in lipases. The gene encoding a lipase from Rhizopus oryzae was cloned and expressed in Escherichia coli. Site-directed mutagenesis of this gene was used to test the validity of computer-aided predictions of the functional roles of specific amino acids in this enzyme. Examination of the kinetic constants of the Rhizopus oryzae lipase variants allowed us to identify amino acid residues which are directly involved in the catalytic reaction or which stabilize the active geometry of the enzyme. The combination of these results with molecular mechanics simulations, based on a homology-derived structural model, provided new information about structure-function relationships. The interpretation of the data is consistent with results obtained with other hydrolases, such as proteases.
Beer
H D
HD
Department of Enzymology, National Biotechnology, Research Centre (GBF), Braunschweig, Germany.
Wohlfahrt
G
G
McCarthy
J E
JE
Schomburg
D
D
Schmid
R D
RD
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D003198:Computer Simulation; D017076:Computer-Aided Design; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012233:Rhizopus / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013045:Species Specificity
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D008049:Lipase