Kinetic and Structural Basis for Acyl-Group Selectivity and NAD(+) Dependence in Sirtuin-Catalyzed Deacylation.
Kinetic and Structural Basis for Acyl-Group Selectivity and NAD(+) Dependence in Sirtuin-Catalyzed Deacylation.
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DOI：
10.1021/acs.biochem.5b00150
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发表时间：
2015-05-19
期刊：
影响因子：
2.9
通讯作者：
Denu, John M.
中科院分区：
文献类型：
--
作者：
Feldman, Jessica L.;Dittenhafer-Reed, Kristin E.;Kudo, Norio;Thelen, Julie N.;Ito, Akihiro;Yoshida, Minoru;Denu, John M.
赖氨酸的酰化是一种重要的蛋白质修饰，可调节多种生物过程。最近研究表明，人类沉默调节蛋白（Sirtuin）家族成员除了具有众所周知的烟酰胺腺嘌呤二核苷酸（NAD⁺）依赖性去乙酰化活性外，还能够催化长链去酰化反应。在此，我们提供了详细的动力学和结构分析，描述了一系列不同的人类沉默调节蛋白（SIRT1、SIRT2、SIRT3和SIRT6）对NAD⁺的依赖性以及酰基长度之间的相互关系。稳态和快速淬灭动力学分析表明，NAD⁺饱和度以及对烟酰胺抑制的敏感性差异反映了每种沉默调节蛋白所表现出的独特动力学行为，并且取决于酰基底物链的长度。尽管2′ - 羟基对C1′ - O - 烷基脒中间体的亲核攻击速率随酰基底物链长度而变化，但这一步骤对于SIRT2和SIRT3仍然是速率决定步骤；然而对于SIRT6，对于长链底物，这一步骤不再是速率限制步骤。SIRT2与十四酰化肽段和NAD⁺共结晶，得到了含有反应产物2′ - O - 十四酰 - 腺苷二磷酸 - 核糖的共复合物结构，揭示了一个潜在的疏水腔以容纳长链酰基，并提出了长链去酰化的一般机制。比较两个分别解析的含有十四酰化肽段或2′ - O - 十四酰 - 腺苷二磷酸 - 核糖的共复合物结构表明，在十四酰 - 核糖连接处存在构象变化，而酶活性位点的结构差异极小。在去酰化反应过程中，脂肪酰基处于相对固定的位置。我们描述了一个动力学和结构模型，以解释各种沉默调节蛋白如何表现出独特的酰基底物偏好以及不同的反应动力学如何影响对NAD⁺的依赖性。并对其生物学意义进行了讨论。
Acylation of lysine is an important protein modification regulating diverse biological processes. It was recently demonstrated that members of the human Sirtuin family are capable of catalyzing long-chain deacylation, in addition to the well-known NAD+-dependent deacetylation activity. Here we provide a detailed kinetic and structural analysis that describes the interdependence of NAD+ and acyl-group length for a diverse series of human Sirtuins, SIRT1, SIRT2, SIRT3 and SIRT6. Steady-state and rapid-quench kinetic analyses indicated that differences in NAD+ saturation and susceptibility to nicotinamide inhibition reflect unique kinetic behavior displayed by each Sirtuin and depend on acyl-substrate chain length. Though the rate of nucleophilic attack of the 2′-hydroxyl on the C1′-O-alkylimidate intermediate varies with acyl substrate chain length, this step remains rate-determining for SIRT2 and SIRT3; however for SIRT6, this step is no longer rate-limiting for long-chain substrates. Co-crystallization of SIRT2 with myristoylated peptide and NAD+ yielded a co-complex structure with reaction product 2′-O-myristoyl-ADP-ribose, revealing a latent hydrophobic cavity to accommodate the long chain acyl group, and suggesting a general mechanism for long chain deacylation. Comparing two separately solved co-complex structures containing either a myristoylated peptide or 2′-O-myristoyl-ADP-ribose indicate there are conformational changes at the myristoyl-ribose linkage with minimal structural differences in the enzyme active site. During the deacylation reaction, the fatty acyl group is held in a relatively fixed position. We describe a kinetic and structural model to explain how various Sirtuins display unique acyl-substrate preferences and how different reaction kinetics influence NAD+ dependence. The biological implications are discussed.
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DOI：
10.1006/bbrc.2000.3000
发表时间：
2000-07-05
影响因子：
3.1
作者：
Frye, RA
通讯作者：
Frye, RA
影响因子：
3
作者：
Moniot, Sebastien;Schutkowski, Mike;Steegborn, Clemens
通讯作者：
Steegborn, Clemens
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
3.7
作者：
Fischer F;Gertz M;Suenkel B;Lakshminarasimhan M;Schutkowski M;Steegborn C
通讯作者：
Steegborn C
DOI：
10.1107/s0907444909042073
发表时间：
2010-01
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Chen VB;Arendall WB 3rd;Headd JJ;Keedy DA;Immormino RM;Kapral GJ;Murray LW;Richardson JS;Richardson DC
通讯作者：
Richardson DC