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 (e = 11mM1cm1) and 378nm, shows a molecular mass of 63kDa, and has Km and kcat values of 170lM and 4.8 s1, 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 = 63lM. If incubated for longer times, MDL72,527 yields irreversible inhibition of the enzyme with a half-life of 15min at 100lM MDL72,527. The mMSO catalytic mechanism, investigated by stopped flow, is consistent with a simple four-step kinetic scheme.
Bellelli, A., Cavallo, S., Nicolini, L., Cervelli, M., Bianchi, M., Mariottini, P., et al. (2004). MOUSE SPERMINE OXIDASE: A MODEL OF THE CATALYTIC CYCLE AND ITS INHIBITOR N,N1-BIS(2,3-BUTADIENYL)-1,4-BUTANEDIAMINE. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 322, 1-8 [10.1016/j.bbrc.2004.07.074].
MOUSE SPERMINE OXIDASE: A MODEL OF THE CATALYTIC CYCLE AND ITS INHIBITOR N,N1-BIS(2,3-BUTADIENYL)-1,4-BUTANEDIAMINE
CERVELLI, MANUELA;MARIOTTINI, Paolo;
2004-01-01
Abstract
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 (e = 11mM1cm1) and 378nm, shows a molecular mass of 63kDa, and has Km and kcat values of 170lM and 4.8 s1, 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 = 63lM. If incubated for longer times, MDL72,527 yields irreversible inhibition of the enzyme with a half-life of 15min at 100lM MDL72,527. The mMSO catalytic mechanism, investigated by stopped flow, is consistent with a simple four-step kinetic scheme.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
