TAILIEUCHUNG - Báo cáo khoa học: Two L-amino acid oxidase isoenzymes from Russell’s viper (Daboia russelli russelli) venom with different mechanisms of inhibition by substrate analogs

Two isoforms, L1and L2,ofl-amino acid oxidase have been isolated from Russell’s viper venom by Sephadex G-100 gel filtration followed by CM-Sephadex C-50 ion exchange chromatography. The enzymes, with different isoelectric points, are monomers of 60–63 kDa as observed from size exclu-sion HPLC and SDS⁄PAGE. | ễFEBS Journal Two L-amino acid oxidase isoenzymes from Russell s viper Daboia russelli russelli venom with different mechanisms of inhibition by substrate analogs Somnath Mandal and Debasish Bhattacharyya Division of StructuralBiology and Bioinformatics Indian Institute of ChemicalBiology Kolkata India Keywords enzyme kinetics inhibitor cross-competition flavoenzyme L-amino acid oxidase mixed inhibition Correspondence D. Bhattacharyya Division of Structural Biology and Bioinformatics Indian Institute of ChemicalBiology Kolkata 700032 India Fax 91 33 2473 5197 0284 Tel 91 33 2473 3491 x 164 E-mail debasish@ Received 1 January 2008 revised 25 February 2008 accepted 27 February 2008 doi Two isoforms Li and L2 of L-amino acid oxidase have been isolated from Russell s viper venom by Sephadex G-100 gel filtration followed by CM-Sephadex C-50 ion exchange chromatography. The enzymes with different isoelectric points are monomers of 60-63 kDa as observed from size exclusion HPLC and SDS PAGE. Partial N-terminal amino acid sequencing of L1 and L2 showed significant homology with other snake venom L-amino acid oxidases. Both the enzymes exhibit marked substrate preference for hydrophobic amino acids maximum catalytic efficiency being observed with L-Phe. Inhibition of L1 and L2 by the substrate analogs N-acetyltry-ptophan and N-acetyl-L-tryptophan amide has been followed. The initial uncompetitive inhibition of L1 followed by mixed inhibition at higher concentrations suggested the existence of two different inhibitor-binding sites distinct from the substrate-binding site. In the case of L2 initial linear competitive inhibition followed by mixed inhibition suggested the existence of two nonoverlapping inhibitor-binding sites one of which is the substratebinding site. An inhibition kinetic study with O-aminobenzoic acid a mimicking substrate with amino carboxylate and hydrophobic parts indicated the presence of three and two binding

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