Department of Pharmaceutical Sciences, Albany College of Pharmacy and Health Sciences, Colchester, VT 05446, USA.
Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23219, USA.
Biomolecules. 2021 Feb 18;11(2):312. doi: 10.3390/biom11020312.
SIRT1 is the most extensively studied human sirtuin with a broad spectrum of endogenous targets. It has been implicated in the regulation of a myriad of cellular events, such as gene transcription, mitochondria biogenesis, insulin secretion as well as glucose and lipid metabolism. From a mechanistic perspective, nicotinamide (NAM), a byproduct of a sirtuin-catalyzed reaction, reverses a reaction intermediate to regenerate NAD through "base exchange", leading to the inhibition of the forward deacetylation. NAM has been suggested as a universal sirtuin negative regulator. Sirtuins have evolved different strategies in response to NAM regulation. Here, we report the detailed kinetic analysis of SIRT1-catalyzed reactions using endogenous substrate-based synthetic peptides. A novel substrate-dependent sensitivity of SIRT1 to NAM inhibition was observed. Additionally, SIRT1 demonstrated pH-dependent deacetylation with normal solvent isotope effects (SIEs), consistent with proton transfer in the rate-limiting step. Base exchange, in contrast, was insensitive to pH changes with no apparent SIEs, indicative of lack of proton transfer in the rate-limiting step. Consequently, NAM inhibition was attenuated at a high pH in proteated buffers. Our study provides new evidence for "activation by de-repression" as an effective sirtuin activation strategy.
SIRT1 是研究最为广泛的人类沉默调节蛋白，具有广泛的内源性靶标。它参与了许多细胞事件的调节，如基因转录、线粒体生物发生、胰岛素分泌以及葡萄糖和脂质代谢。从机制的角度来看，烟酰胺（NAM）是一种 SIRT 催化反应的副产物，通过“碱基交换”逆转反应中间体，从而再生 NAD，导致向前去乙酰化的抑制。NAM 被认为是一种通用的 SIRT 负调节剂。SIRT 已进化出不同的策略来应对 NAM 的调节。在这里，我们使用内源性基于底物的合成肽报告了 SIRT1 催化反应的详细动力学分析。观察到 SIRT1 对 NAM 抑制的新型底物依赖性敏感性。此外，SIRT1 表现出 pH 依赖性去乙酰化，具有正常溶剂同位素效应（SIE），与限速步骤中的质子转移一致。相比之下，碱基交换对 pH 变化不敏感，没有明显的 SIE，表明限速步骤中没有质子转移。因此，在保护缓冲液中高 pH 时，NAM 抑制作用减弱。我们的研究为“去抑制激活”作为一种有效的 SIRT 激活策略提供了新的证据。
