Relationship between conserved consensus site residues and the productive conformation for the TPQ cofactor in a copper-containing amine oxidase fromyeast

A highly conserved asparagine residue is contained in the consensus site se quences of all known copper-containing amine oxidases (CAOs). On the basis of published crystallographic structures, the asparagine is found to re sid e proximal to the active site redox cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ). In this study, the conserved asparagine was changed to an a lanine in a CAO from Hansenula polymorpha expressed in Saccharomyces cerevi siae, and the mutant's catalytic properties were characterized using steady -state kinetics and resonance Raman spectroscopy. Several lines of evidence point to TPQ exisiting in an nonproductive orientation in the mutant, incl uding reductions in several steady-state parameters and an accumulation of an inactive product Schiff base complex when the enzyme is incubated with m ethylamine as the substrate. This product Schiff base complex was previousl y found to form following mutation of another conserved consensus site resi due, a glutamate (or aspartate) at the C + 1 position from TPQ [Cai, D., Do ve, J., Nakamura, N., Sanders-Loehr, J., and Klinman, J. P. (1997) Biochemi stry 36, 11472-11478]. The results suggest that these two residues are cruc ial in maintaining the balance of cofactor mobility versus rigidity expecte d to be necessary during the dual processes of biogenesis and catalysis, re spectively, that all CAOs must accomplish. In addition, a previously uniden tified structural linkage between these two highly conserved residues is pr oposed which spans both subunits of the dimeric CAOs, and may have implicat ions for intersubunit communication.