Investigation on the Structure of the Active Site of Monoamine Oxidase-B by Affinity Labeling with the Selective Inhibitor Lazabemide and by Site-Directed Mutagenesis
| Autor: | Cesura Andrea, Mosé Da Prada, Urs Röthlisberger, Hans-Werner Lahm, Rene Imhof, J. Gottowik, Gabrielle Lang, Pari Malherbe |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Monoamine Oxidase Inhibitors
Stereochemistry Monoamine oxidase Molecular Sequence Data Peptide Flavin group Kidney Transfection Peptide Mapping Polymerase Chain Reaction Biochemistry Cell Line chemistry.chemical_compound Humans Amino Acid Sequence Cysteine Picolinic Acids Site-directed mutagenesis Monoamine Oxidase Chromatography High Pressure Liquid DNA Primers chemistry.chemical_classification Binding Sites Affinity labeling Base Sequence biology Active site Affinity Labels Valine Peptide Fragments Recombinant Proteins Isoenzymes Kinetics chemistry Spectrophotometry Mutagenesis Site-Directed biology.protein Lazabemide Monoamine oxidase B |
| Zdroj: | European Journal of Biochemistry. 236:996-1002 |
| ISSN: | 1432-1033 0014-2956 |
| DOI: | 10.1111/j.1432-1033.1996.00996.x |
| Popis: | The structural features of the active site of human monoamine oxidase B (MAO-B) were investigated by affinity labeling and site-directed mutagenesis. The pseudosubstrate inhibitor N-[2-aminoethyl]-5-chloro-2-pyridine carboxamide HCl (lazabemide) can be irreversibly linked to MAO-B by reduction of the enzyme-inhibitor complex with NaBH(3)CN. Analysis of the flavin spectrum of [(3)H]lazabemide-labeled human MAO-B indicated that insertion of the inhibitor did not occur into the isoalloxazine ring of FAD. After trypsin digestion and HPLC peptide mapping of the radiolabeled enzyme, two labeled peptides were observed. Sequence analysis showed that both peptides started at Val371 of human MAO-B. These results indicate that [(3)H]lazabemide is incorporated into the MAO-B peptide stretch containing the FAD-modified Cys397. The function of putative active-site residues contained in this region was investigated by site-directed mutagenesis and expression of the mutant proteins in HEK-293 cells. Substitution of His382 of MAO-B with an Arg greatly reduced the enzymic activity, suggesting that this residue may represent a nucleophile relevant for the MAO-B catalytic mechanism. Whereas it has been shown that mutation of Cys389 with a Ser residue does not markedly affect the activity of the enzyme [Wu, H.-F., Chen, K. and Shih, J.C. (1993) Mol. Pharmacol. 43, 888-893] the mutant carrying an Ala at this position was virtually inactive. Conversely, substitution of Lys386 (to Met) and Ser394 (to Ala) did not markedly modify the kinetic properties of the enzyme. We also report that mutation of MAO-B Thr158 (to Ala) resulted in a dramatic loss of enzymic activity. [on SciFinder (R)] |
| Databáze: | OpenAIRE |
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