Relationship between conserved consensus site residues and the productive conformation for the TPQ cofactor in a copper-containing amine oxidase from yeast.
A highly conserved asparagine residue is contained in the consensus site sequences of all known copper-containing amine oxidases (CAOs). On the basis of published crystallographic structures, the asparagine is found to reside proximal to the active site redox cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ). In this study, the conserved asparagine was changed to an alanine in a CAO from Hansenula polymorpha expressed in Saccharomyces cerevisiae, 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, including reductions in several steady-state parameters and an accumulation of an inactive product Schiff base complex when the enzyme is incubated with methylamine as the substrate. This product Schiff base complex was previously found to form following mutation of another conserved consensus site residue, a glutamate (or aspartate) at the C + 1 position from TPQ [Cai, D., Dove, J., Nakamura, N., Sanders-Loehr, J., and Klinman, J. P. (1997) Biochemistry 36, 11472-11478]. The results suggest that these two residues are crucial in maintaining the balance of cofactor mobility versus rigidity expected to be necessary during the dual processes of biogenesis and catalysis, respectively, that all CAOs must accomplish. In addition, a previously unidentified structural linkage between these two highly conserved residues is proposed which spans both subunits of the dimeric CAOs, and may have implications for intersubunit communication.
Schwartz
B
B
Department of Chemistry, University of California, Berkeley 94720, USA.
Green
E L
EL
Sanders-Loehr
J
J
Klinman
J P
JP
D000409:Alanine / Q000235:genetics; D006631:Amine Oxidase (Copper-Containing) / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence / Q000235:genetics; D001216:Asparagine / Q000235:genetics; D002384:Catalysis; D003067:Coenzymes / Q000096:biosynthesis / Q000737:chemistry*; D017124:Conserved Sequence / Q000235:genetics; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D007700:Kinetics; D008744:Methylamines / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
C086758:2,4,5-trihydroxyphenylalanine quinone; D003067:Coenzymes; D008744:Methylamines; D004295:Dihydroxyphenylalanine; D001216:Asparagine; C027451:methylamine; D006631:Amine Oxidase (Copper-Containing); D000409:Alanine