HALF-OF-THE-SITES REACTIVITY OF BOVINE SERUM AMINE OXIDASE - REACTIVITY AND CHEMICAL IDENTITY OF THE 2ND SITE

The organic cofactor of bovine serum amine oxidase was identified as 2 ,4,5-trihydroxyphenylalanine quinone by means of the phenylhydrazine a dduct [Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltb y, D., Burligame, A. L. & Klinman, J. P. (1990) Science 248, 981-987]. A still debated question is, however, whether the dimeric protein bin ds two mol phenylhydrazine/mole or only one, that is whether it actual ly contains two identical independent carbonyl cofactors. This matter is addressed in the present study by means of the protein reactions wi th phenylhydrazine and other inhibitors such as semicarbazide and p-py ridine-2-yl-phenylacetohydrazide. The two latter reagents were found t o bind in two steps, one mole/mole dimer in the first step with loss o f catalytic activity but only about (0.10-0.35 mol/mol) in the second one. Similar results were obtained by either optical spectroscopy or b y reverse-phase HPLC of the labelled peptides produced on proteolysis. Irrespective of the inhibitor nature and reacted amount, all adducts formed on proteolysis a single labelled peptide, of same 25-amino-acid composition, showing that the same cofactor is present in both subuni ts, in the same stretch of the polypeptide chain. The slow reaction of the second cofactor may be related to slow conformational equilibria, which-are established after the first cofactor has reacted and are pr obably mediated by a change of the hydrogen bond pattern. The conforme rs spectroscopic properties suggest that they differ in whether the co factor does or does not directly interact with copper.