Half-of-the-sites reactivity of bovine serum amine oxidase. Reactivity and chemical identity of the second site.
The organic cofactor of bovine serum amine oxidase was identified as 2,4,5-trihydroxyphenylalanine quinone by means of the phenylhydrazine adduct [Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burligame, A.L. & Klinman, J.P. (1990) Science 248, 981-987]. A still debated question is, however, whether the dimeric protein binds two mol phenylhydrazine/mole or only one, that is whether it actually contains two identical independent carbonyl cofactors. This matter is addressed in the present study by means of the protein reactions with phenylhydrazine and other inhibitors such as semicarbazide and p-pyridine-2-yl-phenylacetohydrazide. The two latter reagents were found to bind in two steps, one mole/mole dimer in the first step with loss of catalytic activity but only about (0.10-0.35 mol/mol) in the second one. Similar results were obtained by either optical spectroscopy or by 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 subunits, 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 probably mediated by a change of the hydrogen bond pattern. The conformers spectroscopic properties suggest that they differ in whether the cofactor does or does not directly interact with copper.
De Biase
D
D
Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Università La Sapienza, Roma, Italy.
Agostinelli
E
E
De Matteis
G
G
Mondovì
B
B
Morpurgo
L
L
D006631:Amine Oxidase (Copper-Containing); D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006868:Hydrolysis; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000097:blood* / Q000737:chemistry; D011489:Protein Denaturation; D013379:Substrate Specificity
D006631:Amine Oxidase (Copper-Containing); D000587:Oxidoreductases Acting on CH-NH Group Donors