title: Mutation of a strictly conserved, active-site residue alters substrate specificity and cofactor biogenesis in a copper amine oxidase.
abstract: The copper amine oxidases (CAOs) catalyze both the single-turnover modification of a peptidyl tyrosine to form the active-site cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ) and the oxidative deamination of primary amines using TPQ. The function of a strictly conserved tyrosine located within hydrogen-bonding distance to TPQ has been explored by employing site-directed mutagenesis on the enzyme from H. polymorpha to form the mutants Y305A, Y305C, and Y305F. Both Y305A and Y305C behave similarly with regard to aliphatic amine oxidase activity, showing 3-7-fold decreases in kinetic parameters relative to WT, while the more conservative substitution of Y305F results in a >100-fold decrease in kcat and >500-fold decrease in kcat/Km relative to WT for the reductive half-reaction. The oxidation of benzylamine by all three mutants is severely impaired, with very significant effects seen in the oxidative half-reaction. CAO activity was studied as a function of pH for WT and Y305A proteins. Profiles for WT-catalyzed methylamine oxidation and Y305A-catalyzed ethylamine oxidation are comparable, while profiles of Y305A-catalyzed methylamine oxidation suggest the pH-dependent build-up of an inhibitory intermediate, which was subsequently observed spectrophotometrically and is attributed to the product Schiff base. The relative effects of mutations at Y305 on catalytic turnover are, thus, concluded to be dependent on the nature of the amino acid which substitutes for tyrosine and the substrate used in amine oxidase assays. TPQ biogenesis experiments demonstrate a approximately 800-fold decrease in kobs for apo-Y305A compared to WT. Despite the strict conservation of Tyr305 in all CAOs, neither biogenesis nor catalytic turnover is abolished upon mutation of this residue. We propose an important, but nonessential, role for Tyr305 in the positioning of the TPQ precursor for biogenesis, and in the maintenance of the correct conformation for TPQ-derived intermediates during catalytic turnover.
authors: Department of Chemistry and Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
authors: J M
authors: JM
authors: Hevel
authors: S A
authors: SA
authors: Mills
authors: J P
authors: JP
authors: Klinman
mesh_terms: D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010843:Pichia / Q000201:enzymology; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012545:Schiff Bases / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: C086758:2,4,5-trihydroxyphenylalanine quinone; D012545:Schiff Bases; D004295:Dihydroxyphenylalanine; D006631:Amine Oxidase (Copper-Containing)