An unexpected role for the active site base in cofactor orientation and flexibility in the copper amine oxidase from Hansenula polymorpha

The role of the active site aspartate base in the aminotransferase mechanis m of the copper amine oxidase from the yeast Hansenula polymorpha has been probed by site-directed mutagenesis. The D319E mutant catalyzes the oxidati on of methylamine and phenethylamine, but not that of benzylamine. k(cat)/K -m for methylamine is found to be 80-fold reduced compared to that of the w ild type. Viscosogen and substrate and solvent deuteration have no effect o n this parameter for D319E, which is suggestive of limitation of k(cat)/K-m by a conformational change. This conformational change is proposed to be t he movement of the cofactor into a productive orientation upon the binding of substrate. In the absence of substrate, a flipped cofactor orientation i s likely, on the basis of resonance Raman evidence that the C5 carbonyl of the cofactor is less solvent accessible than the C3 hydrogen. k(cat) for D3 19E methylamine oxidase is reduced 200-fold compared to that of the wild ty pe and is unaffected by substrate deuteration, but displays a substantial s olvent isotope effect. A 428 nm absorbance is evident under conditions of s aturating methylamine and oxygen with D319E. The D319N mutant is observed t o produce a similar absorbance at 430 nm when treated with ammonia despite the fact that this mutant has: no amine oxidase activity. Resonance Raman s pectroscopy indicates the formation of a covalent ammonia adduct and identi fies it as the deprotonated iminoquinone. In contrast, when the D319E mutan t is reacted with ammonia, it gives predominantly a 340-350 nm species. Thi s absorbance is ascribed to a localization of the cofactor oxyanion induced by binding of the cation at the active site and not to covalent adduct for mation. Resonance Raman spectroscopic examination of the steady state speci es of D319E methylamine oxidation, in combination with the kinetic data, in dicates that the 428 nm species is the deprotonated iminoquinone produced u pon reoxidation of the reduced cofactor. A model is proposed in which a cen tral role of the active site base is to position the free cofactor and seve ral enzyme intermediates for optimal activity.