C-终端β8-α9相互作用调节热稳定性和酶活性在高热友雌激酶EstE1和中热友雌激酶rPPE中不同
Tu Anh Nguyen1, Sei-Heon Jang1, ChangWoo Lee1
1Department of Biomedical Science and Center for Bio-Nanomaterials, Daegu University, Gyeongsan, South Korea.
Applied and environmental microbiology
|June 8, 2023
概括
这项研究揭示了酶如何使用疏水相互作用或键来保持稳定性和活性. 虽然这两种相互作用都同样有助于热稳定,但键增强了酶中的催化活性.
科学领域:
- 生物化学 生物化学
- 蛋白质科学 蛋白质科学
- 酶学 是一种酶学.
背景情况:
- 非共价相互作用,包括疏水相互作用和键,对于蛋白质的折叠和稳定性至关重要.
- 这些相互作用在α/β-水解酶,特别是酶中在不同环境中的特定作用仍然不完全理解.
- 雌激酶是重要的酶,能水解雌激酶键,并在连接C终端β8链和α9螺旋的循环中具有催化性胺残留物.
研究的目的:
- 为了研究超热性雌激酶EstE1和中热性雌激酶rPPE如何利用疏水性相互作用和键适应不同温度.
- 阐明这些雌激酶稳定β8-α9链螺旋的独特机制.
- 确定疏水相互作用和键对这些酶的热稳定性和催化活性的差异性贡献.
主要方法:
- 在不同的环境条件下对高热性雌激酶 (EstE1) 和中热性雌激酶 (rPPE) 的比较分析.
- 位点定向的突变发生改变特定的氨基酸残留物,这些残留物参与β8-α9链螺旋内的疏水相互作用和键.
- 使用差分扫描热量计或循环二元化等技术评估热稳定性.
- 酶活性测定用于量化催化效率.
主要成果:
- 超热性雌激酶EstE1,在其二次形式,通过疏水相互作用 (Phe276和Leu299) 稳定β8-α9链螺旋,形成一个闭合的二次接口.
- 在其单体形式的 mesophilic esterase rPPE 中,通过键 (Tyr281 和 Gln306) 稳定了相同的链螺旋.
- 破坏这些相互作用的突变降低了热稳定性,而EstE1中的工程键和rPPE中的疏水性相互作用产生了与野生类型对应物相比的热稳定性.
- 重要的是,具有β8-α9键 (EstE1 (F276Y/L299Q) 和rPPE WT) 的变体表现出明显更高的酶活性,而不是那些具有疏水性相互作用的变体,这归因于催化循环的灵活性增加.
结论:
- 疏水相互作用和键均对α/β-水解酶的热稳定性有着同等的贡献.
- β8-α9键有利于增强催化活性,可能是由于催化循环的灵活性增加.
- 这些发现突显了酶对各种环境的适应策略,并为具有定制稳定性和活性的酶的蛋白质工程提供了洞察力.
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