An investigation of bovine serum amine oxidase active site stoichiometry: evidence for an aminotransferase mechanism involving two carbonyl cofactors per enzyme dimer.
Recent evidence has shown that the active site cofactor in bovine serum amine oxidase (BSAO) is 2,4,5-trihydroxyphenylalanine or 6-hydroxydopa [Janes et al. (1990) Science 248, 981]. However, much ambiguity remains regarding the mechanism of the enzymatic reaction. Conflicting data exist for both the number of functional active sites in the dimeric enzyme and for the oxygen dependence of product release. To resolve these questions, a new method has been developed for the purification of BSAO which leads to the isolation of specific activity greater than or equal to 0.4 unit/mg of enzyme in 2-3 weeks. This highly active enzyme has been used to quantitate both aldehyde and ammonia release in the reductive half-reaction. Anaerobic incubation of enzyme and substrate resulted in the production of 2 mol of aldehyde/mol of enzyme, indicating the presence of a cofactor at each enzyme subunit. As anticipated for an aminotransferase reaction, no ammonia release was detected under comparable conditions. Active site titration of enzyme samples of varying specific activity with phenylhydrazine extrapolates to 1 mol of inhibitor/mol of enzyme subunit for BSAO of specific activity = 0.48 unit/mg. These findings contrast with numerous, previous reports of only one functional cofactor per enzyme dimer in copper amine oxidases.
Janes
S M
SM
Department of Chemistry, University of California, Berkeley 94720.
Klinman
J P
JP
D006631:Amine Oxidase (Copper-Containing); D000693:Anaerobiosis; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000097:blood / Q000378:metabolism*; D010659:Phenylhydrazines / Q000494:pharmacology; D000637:Transaminases / Q000378:metabolism*
D010659:Phenylhydrazines; C030299:phenylhydrazine; D006631:Amine Oxidase (Copper-Containing); D000587:Oxidoreductases Acting on CH-NH Group Donors; D000637:Transaminases