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 (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.
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
Andrea
A
Bellelli
Stefano
S
Cavallo
Laura
L
Nicolini
Manuela
M
Cervelli
Marzia
M
Bianchi
Paolo
P
Mariottini
Massimo
M
Zelli
Rodolfo
R
Federico
D000818:Animals; D002384:Catalysis; D003198:Computer Simulation; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007527:Isoenzymes; D007700:Kinetics; D051379:Mice; D008956:Models, Chemical; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000037:antagonists & inhibitors / Q000737:chemistry*; D015202:Protein Engineering; D011700:Putrescine / Q000031:analogs & derivatives* / Q000737:chemistry*; D013379:Substrate Specificity; D000097667:Polyamine Oxidase
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011700:Putrescine; D000097667:Polyamine Oxidase; C044445:MDL 72527