Comparative mechanistic and structural approaches to investigate the membrane-bound enzymes monoamine oxidase A and monoamine oxidase B

Comparative mechanistic and structural studies of two mitochondrial outer membrane bound enzymes: monoamine oxidases A and B (MAO A and MAO B) are described in this dissertation. The kinetic and structural biology studies on MAO A and MAO B are greatly facilitated by the initial results in this dissertation on the high-level expression of recombinant human liver MAO A in Pichia pastoris which demonstrate significant advantages in the heterologous expression as compared with the previous S. cerevisiae system. A series of substrate analogues were synthesized with stereospecific 2H labeling at the alpha-carbons. Only the pro-R hydrogen is removed on catalytic oxidation by either MAO A or MAO B. Spectral and kinetic studies of pyridylalkylamine analogues show no evidence for the intermediate formation of a flavin C4a adduct on catalytic turnover by either enzyme. Mechanistic studies of MAO inhibition by arylalkylhydrazines suggest the inhibiting species to be an arylalkyl radical produced by O2 oxidation of the diazene product of enzyme catalysis. The main focus of this dissertation is to understand the functional role of the "aromatic cage" in MAO that consists of two tyrosyl residues that occupying conformations approximately perpendicular to the re side of the flavin ring. Structural and catalytic studies of four Y435 (MAO B) and Y444 (MAO A) mutant proteins suggest the functional role of this cage is to polarize the substrate amine nitrogen to become a better nucleophile in catalysis and possibly to deprotonate the amine moiety prior to substrate oxidation by the flavin coenzyme. The results from this dissertation support the proposed polar nucleophilic mechanism for MAO catalysis and provide insights for future mechanistic and structural studies of MAO A and MAO B.