Mouse spermine oxidase: a model of the catalytic cycle and its inhibition by N,N1-bis(2,3-butadienyl)-1,4-butanediamine |
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Authors: | Bellelli Andrea Cavallo Stefano Nicolini Laura Cervelli Manuela Bianchi Marzia Mariottini Paolo Zelli Massimo Federico Rodolfo |
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Affiliation: | Dipartimento di Scienze Biochimiche Alessandro Rossi Fanelli, Università di Roma La Sapienza and Istituto di Biologia e Patologia Molecolari del CNR, I-00185 Rome, Italy. andrea.bellelli@uniroma1.it |
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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 (epsilon=11mM(-1)cm(-1)) and 378nm, shows a molecular mass of approximately 63kDa, and has K(m) and k(cat) values of 170microM and 4.8s(-1), using spermine as substrate; it is unable to oxidize other free or acetylated polyamines. The mechanism-based PAO inhibitor N,N(1)-bis(2,3-butadienyl)-1,4-butanediamine (MDL72,527) acts as a competitive inhibitor of mSMO, with an apparent dissociation constant K(i)=63microM. If incubated for longer times, MDL72,527 yields irreversible inhibition of the enzyme with a half-life of 15min at 100microM MDL72,527. The mMSO catalytic mechanism, investigated by stopped flow, is consistent with a simple four-step kinetic scheme. |
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Keywords: | Polyamine oxidase Spermine oxidase Mouse Protein engineering Stopped flow |
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