Mouse spermine oxidase: a model of the catalytic cycle and its inhibition by N,N1-bis(2,3-butadienyl)-1,4-butanediamine

Spermine oxidase (SMO) is a recently described flavoenzyme belonging to the class of polyamine oxidases (PAOs) and participating in the polyamine metabolism in animal cells. In this paper we describe the expression, purification, and characterization of the catalytic properties of a recombinant mouse SMO (mSMO). The purified enzyme has absorbance peaks at 457nm (ε=11mM−1cm−1) and 378nm, shows a molecular mass of ∼63kDa, and has Km and kcat values of 170μM and 4.8s−1, using spermine as substrate; it is unable to oxidize other free or acetylated polyamines. The mechanism-based PAO inhibitor N,N1-bis(2,3-butadienyl)-1,4-butanediamine (MDL72,527) acts as a competitive inhibitor of mSMO, with an apparent dissociation constant Ki=63μM. If incubated for longer times, MDL72,527 yields irreversible inhibition of the enzyme with a half-life of 15min at 100μM MDL72,527. The mMSO catalytic mechanism, investigated by stopped flow, is consistent with a simple four-step kinetic scheme. [Copyright &y& Elsevier]