Mechanism of nicotinamide inhibition and transglycosidation by Sir2 histone/protein deacetylases
Mechanism of nicotinamide inhibition and transglycosidation by Sir2 histone/protein deacetylases
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DOI：
10.1074/jbc.m306552200
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发表时间：
2003-12-19
影响因子：
4.8
通讯作者：
Denu, JM
中科院分区：
文献类型：
--
作者：
Jackson, MD;Schmidt, MT;Denu, JM
沉默信息调节因子2（Sir 2）酶催化NAD（+）依赖性蛋白质/组蛋白脱乙酰化，其中乙酰基从赖氨酸β-氨基转移到NAD（+）的ADP-核糖部分，产生烟酰胺和新的代谢物O-乙酰基-ADP核糖. Sir 2蛋白已被证明可以调节基因沉默、代谢酶和寿命。最近，烟酰胺被认为是细胞Sir 2功能的直接负调节剂;然而，烟酰胺抑制的机制尚未建立。Sir 2酶是多功能的，因为脱乙酰酶反应涉及烟酰胺-核糖基的裂解、酰胺键的裂解和乙酰基最终转移到ADP-核糖的2 ′-核糖羟基。在这里，我们证明了烟酰胺抑制是烟酰胺拦截ADP-核糖基酶-乙酰肽中间体并再生NAD（+）（转糖苷作用）的结果。细胞的影响进行了讨论。发现多种3-取代吡啶是酶催化转糖苷作用的底物。数据的布朗斯台德图产生了+0.98的斜率，与过渡态中几乎完全的正电荷的发展一致，并且与攻击亲核试剂的碱性作为反应性的强预测因子一致。NAD（+）类似物包括β-2 '-脱氧-2'-氟核糖-NAD（+）和His-至- Ala突变体用于探测烟酰胺-核糖基切割和乙酰基转移的机制。我们证明烟酰胺-核糖基裂解不同于乙酰基转移到2 '-OH核糖。所观察到的使用β-2 ′-脱氧-2 ′-氟代核糖- NAD（+）的不稳定的1 ′-乙酰化- ADP-氟代核糖中间体的酶催化形成支持了这样的机理，其中在烟酰胺-核糖基裂解后，乙酰化底物的羰基氧攻击C-1 ′核糖以形成初始的亚胺鎓加合物。
Silent information regulator 2 ( Sir2) enzymes catalyze NAD(+)- dependent protein/ histone deacetylation, where the acetyl group from the lysine epsilon- amino group is transferred to the ADP- ribose moiety of NAD(+), producing nicotinamide and the novel metabolite O- acetyl- ADPribose. Sir2 proteins have been shown to regulate gene silencing, metabolic enzymes, and life span. Recently, nicotinamide has been implicated as a direct negative regulator of cellular Sir2 function; however, the mechanism of nicotinamide inhibition was not established. Sir2 enzymes are multifunctional in that the deacetylase reaction involves the cleavage of the nicotinamide- ribosyl, cleavage of an amide bond, and transfer of the acetyl group ultimately to the 2'- ribose hydroxyl of ADP- ribose. Here we demonstrate that nicotinamide inhibition is the result of nicotinamide intercepting an ADP- ribosylenzyme- acetyl peptide intermediate with regeneration of NAD(+) ( transglycosidation). The cellular implications are discussed. A variety of 3- substituted pyridines was found to be substrates for enzyme- catalyzed transglycosidation. A Bronsted plot of the data yielded a slope of + 0.98, consistent with the development of a nearly full positive charge in the transition state, and with basicity of the attacking nucleophile as a strong predictor of reactivity. NAD(+) analogues including beta-2'-deoxy-2'- fluororibo-NAD(+) and a His- to- Ala mutant were used to probe the mechanism of nicotinamide- ribosyl cleavage and acetyl group transfer. We demonstrate that nicotinamide-ribosyl cleavage is distinct from acetyl group transfer to the 2'-OH ribose. The observed enzyme- catalyzed formation of a labile 1'- acetylated- ADP- fluororibose intermediate using beta-2'- deoxy-2'- fluororibo- NAD(+) supports a mechanism where, after nicotinamide- ribosyl cleavage, the carbonyl oxygen of acetylated substrate attacks the C-1' ribose to form an initial iminium adduct.