小鼠精胺氧化酶：催化循环模型及其受N,N1-双(2,3-丁二烯基)-1,4-丁二胺抑制的情况

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

作者信息

Bellelli Andrea, Cavallo Stefano, Nicolini Laura, Cervelli Manuela, Bianchi Marzia, Mariottini Paolo, Zelli Massimo, Federico Rodolfo

机构信息

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.

出版信息

Biochem Biophys Res Commun. 2004 Sep 10;322(1):1-8. doi: 10.1016/j.bbrc.2004.07.074.

DOI:10.1016/j.bbrc.2004.07.074

PMID:15313165

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.

摘要

精胺氧化酶（SMO）是一种最近被描述的黄素酶，属于多胺氧化酶（PAO）类，参与动物细胞中的多胺代谢。在本文中，我们描述了重组小鼠SMO（mSMO）的表达、纯化及其催化特性的表征。纯化后的酶在457nm（ε = 11mM⁻¹cm⁻¹）和378nm处有吸收峰，分子量约为63kDa，以精胺为底物时，Kₘ和kₑₐₜ值分别为170μM和4.8s⁻¹；它不能氧化其他游离或乙酰化的多胺。基于机制的PAO抑制剂N,N⁽¹⁾-双（2,3-丁二烯基）-1,4-丁二胺（MDL72,527）作为mSMO的竞争性抑制剂，表观解离常数Kᵢ = 63μM。如果孵育时间更长，MDL72,527会对该酶产生不可逆抑制，在100μM MDL72,527时半衰期为15分钟。通过停流法研究的mMSO催化机制与简单的四步动力学方案一致。