AN INVESTIGATION OF BOVINE SERUM AMINE OXIDASE ACTIVE-SITE STOICHIOMETRY - EVIDENCE FOR AN AMINOTRANSFERASE MECHANISM INVOLVING 2 CARBONYL COFACTORS PER ENZYME DIMER

被引:129
作者
JANES, SM [1 ]
KLINMAN, JP [1 ]
机构
[1] UNIV CALIF BERKELEY,DEPT CHEM,BERKELEY,CA 94720
关键词
D O I
10.1021/bi00232a034
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:4599 / 4605
页数:7
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