title: Loss of serotonin oxidation as a component of the altered substrate specificity in the Y444F mutant of recombinant human liver MAO A.
abstract: To investigate the roles of tyrosyl residues located near the covalent 8alpha-S-cysteinyl FAD in monoamine oxidase A (MAO A) and to test the suggestion that MAO A and plant polyamine oxidase may have structural homology, tyrosyl to phenylalanyl mutants of MAO A at positions 377, 402, 407, 410, 419, and 444 were constructed and expressed in Saccharomyces cerevisiae. All mutant enzymes were expressed and exhibited lower specific activities as compared to WT MAO A using kynuramine as substrate. The lowest specific activities in this assay are exhibited by the Y407F and Y444F mutant enzymes. On purification and further characterization, these two mutants were found to each contain covalent FAD. Both mutant enzymes are irreversibly inhibited by the MAO A inhibitor clorgyline and exhibit binding stoichiometries of 0.54 (Y407F) and 0.95 (Y444F) as compared to 1.05 for WT MAO A. Y444F MAO A oxidizes kynuramine with a k(cat) <2% of WT enzyme and is greater than 100-fold slower in catalyzing the oxidation of phenylethylamine or of serotonin. In contrast, Y444F MAO A oxidizes p-CF(3)-benzylamine at a rate 25% that of WT enzyme. Steady state and reductive half-reaction stopped-flow data using a series of para-substituted benzylamine analogues show Y444F MAO A exhibits quantitative structure activity relationships (QSAR) properties on analogue binding and rates of substrate oxidation very similar to that exhibited by the WT enzyme (Miller and Edmondson (1999) Biochemistry 38, 13670): log K(d) = -(0.37 +/- ()()0.07)V(W)(x0.1) - 4.5 +/- 0.1; log k(red) = +(2.43 +/- 0.19)sigma + 0.17 +/- 0.05. The Y444F MAO A mutant also exhibits similar QSAR properties on the binding of phenylalkyl side chain amine analogues as WT enzyme: log K(i) = (4.37 +/- 0.51)E(S) + 1.21 +/- 0.77. These data show that mutation of Y444F in MAO A results in a mutant that has lost its ability to efficiently oxidize serotonin (its physiological substrate) but, however, exhibits unaltered quantitative structure-activity parameters in the binding and rate of benzylamine analogues. The mechanism of C-H abstraction is therefore unaltered. The suggestion that polyamine oxidase and monoamine oxidase may have structural homology appears to be valid as regards Y444 in MAO A and Y439 in plant polyamine oxidase.
authors.affiliation: Department of Biochemistry, Emory University, Atlanta, Georgia 30322-3050, USA.
authors.forename: R K
authors.initials: RK
authors.lastname: Nandigama
authors.forename: J R
authors.initials: JR
authors.lastname: Miller
authors.forename: D E
authors.initials: DE
authors.lastname: Edmondson
mesh_terms: D000818:Animals; D001596:Benzylamines / Q000378:metabolism; D003010:Clorgyline / Q000494:pharmacology; D006801:Humans; D007735:Kynuramine / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008297:Male; D015394:Molecular Structure; D008995:Monoamine Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008996:Monoamine Oxidase Inhibitors / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D021281:Quantitative Structure-Activity Relationship; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012701:Serotonin / Q000378:metabolism*; D013379:Substrate Specificity; D000097667:Polyamine Oxidase
chemical_list: D001596:Benzylamines; D003010:Clorgyline; D007735:Kynuramine; D008995:Monoamine Oxidase; D008996:Monoamine Oxidase Inhibitors; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011994:Recombinant Proteins; D012701:Serotonin; D000097667:Polyamine Oxidase; C030796:benzylamine