HALF-SITE REACTIVITY WITH P-NITROPHENYLHYDRAZINE AND SUBUNIT SEPARATION OF THE DIMERIC COPPER-CONTAINING AMINE OXIDASE FROM ASPERGILLUS-NIGER

Structural properties of dimeric (2 x 75 kDa) copper-containing amine oxidase (EC 1.4.3.6) from Aspergillus niger were studied. The enzyme t reated with SDS was dissociated into subunits which showed different m obility on polyacrylamide gel without SDS. The separated subunits had no activity but a quinone moiety was detected in both by a redox-cycli c quinone staining. After titration of the enzyme with p-nitrophenylhy drazine, which showed half-site reactivity (1 mole per dimer), and SDS treatment both p-nitro-phenylhydrazone and a remaining quinone moiety were detected in each subunit. It is suggested that the half-site rea ctivity with phenylhydrazine is caused by conformational changes after binding of the inhibitor to any one of the active sites leading to in accessibility of the second active site for the inhibitor. The differe nce in electrophoretic mobility of the separated subunits originates p robably from their structural difference likely to occur outside the a ctive site, even if the amino acid sequences of the subunits appear to be identical.