COVALENT FLAVIN COENZYMES IN FLAVOENZYME CATALYSIS

对8 α - s -半胱氨酸进行了机理和结构研究

Mechanistic and structural studies are proposed on the 8alpha-S-cysteinyl FAD-containing enzymes: monoamine oxidase B (MAO B) and monoamine oxidase A (MAO A). Human MAO B and MAO A expressed in yeast will be purified and characterized, and compared with the physical and chemical properties of human placental MAO A and bovine liver MAO B. A systematic investigation of ring substituent effects on the binding and rate of CH bond cleavage of benzylamine substrates will be extended from current work on bovine MAO B to recombinant human MAO A and MAO B. Ring-substituent effects on phenethylamine substrate analogs on the binding and rate of CH bond cleavage will also be investigated with MAO A and MAO B and the results compared with the benzylamine data to provide detailed information on steric effects on catalysis.Investigations into whether tunneling contributes to the hydrogen transfer step in MAO B and MAO A will be done in collaboration with Dr. J. Klinman, U. Calif., Berkeley. Evidence for the catalytic involvement and identity of an amino acid radical which may function in hydrogen atom abstraction will be sought in MAO A and MAO B using ESR and ENDOR of native and isotopically labeled enzymes. The oxidation/reduction potentials for the OX/SQ and SQ/HQ couples of the covalent flavin coenzymes in MAO B and in MAO A will be determined using spectrocoulometry. The results of these studies should provide new insights into the catalytic mechanisms of MAO A and of MAO B and provide a basis for the development of specific inhibitors for each enzyme which are of neuropharmacological importance as targets for drug therapy. Anti- depressants used clinically are MAO A inhibitors while the clinical use of MAO B inhibitors is to potentiate L-Dopa therapy of patients with Parkinson's Disease.