Mutation of a strictly conserved, active-site residue alters substrate specificity and cofactor biogenesis in a copper amine oxidase

The copper amine oxidases (CAOs) catalyze both the single-turnover modifica tion of a peptidyl tyrosine to form the active-site cofactor 2,4,5-trihydro xyphenylalanine quinone (TPQ) and the oxidative deamination of primary amin es using TPQ. The function of a strictly conserved tyrosine located within hydrogen-bonding distance to TPQ has been explored by employing site-direct ed mutagenesis on the enzyme from H. polymorpha to form the mutants Y305A, Y305C, and Y305F. Both Y305A and Y305C behave similarly with regard to alip hatic amine oxidase activity, showing 3-7-fold decreases in kinetic paramet ers relative to WT, while the more conservative substitution of Y305F resul ts in a >100-fold decrease in k(cat) and >500-fold decrease in k(cat)/K-m r elative to WT for the reductive half-reaction. The oxidation of benzylamine by all three mutants is severely impaired, with very significant effects s een in the oxidative half-reaction. CAO activity was studied as a function of pH for WT and Y305A proteins. Profiles for WT-catalyzed methylamine oxid ation and Y305A-catalyzed ethylamine oxidation are comparable, while profil es of Y305A-catalyzed methylamine oxidation suggest the pH-dependent buildu p of an inhibitory intermediate, which was subsequently observed spectropho tometrically and is attributed to the product Schiff base. The relative eff ects of mutations at Y305 on catalytic turnover are, thus, concluded to be dependent on the nature of the amino acid which substitutes for tyrosine an d the substrate used in amine oxidase assays. TPQ biogenesis experiments de monstrate a similar to 800-fold decrease in k(obs) for apo-Y305A compared t o WT. Despite the strict conservation of Tyr305 in all CAOs, neither biogen esis 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.