Half‐of‐the‐sites Reactivity of Bovine Serum Amine Oxidase

Abstract

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.

Crystal Structure of Amine Oxidase from Bovine Serum

“…In fact, the end of the arm of one subunit is positioned at the mouth of the active-site channel of the other subunit, suggesting the possibility of interaction between the two active sites. To this regard, in the case of BSAO half-of-thesites reactivity has been reported, 49,50 suggesting the possibility of a negative cooperativity.…”

“…44 However, functional and mechanistic studies on BSAO have yielded limited structural information. 19,[21][22][23]32,[45][46][47][48][49][50][51] As such, our current understanding of this enzyme, including its substrate specificity in terms of structural features, is largely speculative.…”

Copper/topaquinone-containing amine oxidase from lentil seedlings and bovine plasma: Catalytic mechanism and energetic domains

“…for a copper conformational role in the native protein, namely of maintaining a functional conformation at the active site. In addition these studies suggest that while LSAO subunits react independently, in BSAO the two subunits are identical but interdependent in activity [45]. This means that each subunit contains five energetic domains and the binding of the substrate or inhibitors to the active site of each subunit blocks the binding of the above molecules to the active site of the other subunit [7,46].…”

The Role of Protein Crystallography in Defining the Mechanisms of Biogenesis and Catalysis in Copper Amine Oxidase

“…Finally, it is well documented that CAOs function as obligate dimers, and evidence suggests that some, but not all, CAOs exhibit cooperativity through long-range conformational changes propagated through the CAO dimer from one active site to the other [86,114–118]. BSAO and ANAO exhibit half-site reactivity with regard to hydrazine inhibitors [114,115,118].…”

“…BSAO and ANAO exhibit half-site reactivity with regard to hydrazine inhibitors [114,115,118]. In addition, kinetic work using HPAO-1 heterodimers which contain zinc bound in one active site of the HPAO-1 dimer and copper in the second, could not efficiently carry out the oxidative half-reaction at either active site, suggesting that communication between the two metal binding sites influences oxidative chemistry [119].…”