An active site, cofactor-containing peptide has been obtained in high yield from bovine serum amine oxidase. Sequencing of this pentapeptide indicates: Leu-Asn-X-Asp-Tyr. Analysis of the peptide by mass spectrometry, ultraviolet-visible spectroscopy, and proton nuclear magnetic resonance leads to the identification of X as 6-hydroxydopa. This result indicates that, contrary to previous proposals, pyrroloquinoline quinone is not the active site cofactor in mammalian copper amine oxidases. Although 6-hydroxydopa has been implicated in neurotoxicity, the data presented suggest that this compound has a functional role at an enzyme active site.

The nature of the active site cofactor and the amino acid sequence flanking this structure have been determined in a range of copper amine oxidases. For enzymes from porcine plasma, porcine kidney, and pea seedlings, proteolytic digestion was performed on phenylhydrazone or p-nitrophenylhydrazone derivatives. Thermolysin treatment leads to relatively small active site peptides, which have been characterized by Edman degradation and by resonance Raman spectroscopy. Resonance Raman spectra of peptides show identical peak positions and intensities relative to each other and to a model p-nitrophenylhydrazone derivative of topaquinone hydantoin, establishing topaquinone as the cofactor in each instance. Edman degradation of peptides provides active site sequences for comparison to previous determinations with bovine serum and yeast amine oxidases. The available data establish a consensus sequence of Asn, Topa, Asp/Glu. Trypsin leads to significantly longer peptides, which reveal a high degree of sequence identity between plasma proteins from bovine and porcine sources (89%), with significantly decreased identity between the porcine serum and intracellular amine oxidases (56%). A lower degree of identity (45%) is observed between the pea seedling and mammalian enzymes. As an alternative to the isolation of active site peptides for topaquinone identification, visible spectra of intact proteins have been investigated. It is shown that p-nitrophenylhydrazone derivatives of native enzymes, active site-derived peptides, and a topaquinone model exhibit identical behavior, absorbing at 457-463 nm at neutral pH (pH 7.2) and at 575-587 nm in basic solution (1-2 M KOH). These spectral properties, which appear unique to topaquinone, provide a rapid and simple test for the presence of this cofactor in intact enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

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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