用基质3S-hydroxybutanoyl-CoA进行结构酶学研究:双功能MFE1是一种比单功能HAD更低效的脱酶
Shruthi Sridhar1, Tiila-Riikka Kiema2, Werner Schmitz3
1Faculty of Biochemistry and Molecular Medicine, University of Oulu, Finland.
FEBS open bio
|March 8, 2024
概括
多功能酶类型-1 (MFE1) 有两个β-氧化活性位点. 由于其复杂的折叠,老鼠MFE1是一种比人类HSHAD慢的脱酶.
科学领域:
- 生物化学 生化学
- 酶学 是一种酶学.
- 结构生物学 结构生物学
背景情况:
- 多功能酶类型-1 (MFE1) 对于β-氧化至关重要,具有2E--CoA水合酶 (ECH) 和3S---CoA脱酶 (HAD) 活动.
- 了解MFE1的结构功能关系是阐明其在代谢途径中的作用的关键.
研究的目的:
- 研究大鼠MFE1 (RnMFE1)及其变体的结构和酶学特性.
- 为了比较RnMFE1的催化效率与人类的3S-hydroxyacyl-CoA脱酶 (HsHAD).
主要方法:
- 蛋白质结晶学被用来研究ECH活性部位内的结合相互作用.
- 进行了稳定状态前和稳定状态运动实验,以分析HAD活性和基质结合.
- 使用酶变体 (E123A和BCDE) 来探测特定活性位点的贡献.
主要成果:
- 结晶学数据揭示了基质与催化性谷氨酸在ECH活性部位的键相互作用.
- 动力学分析显示RnMFE1和HSHAD的NAD+/NADH结合率相似.
- 与HsHAD.相比,RnMFE1的脱催化率 (kcat,kchem) 大约是HsHAD.的10倍.
结论:
- RnMFE1的ECH活性位点通过结与基质进行接触.
- 虽然RnMFE1和HSHAD表现出类似的辅因子结合,但RnMFE1显示脱酶的催化效率降低.
- 建议RnMFE1的更复杂的折叠是其较慢的脱率的基础,与HSHAD相比.
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