在一个工程化过氧化酶细胞染色体P450变体中的海姆水联结合协调环境的表征
Matthew N Podgorski1, Joel H Z Lee1, Joshua S Harbort2
1Department of Chemistry, University of Adelaide, Adelaide, SA 5005, Australia.
Journal of inorganic biochemistry
|October 14, 2023
概括
突变细胞染色体P450 (CYP) 199A4中的一个关键残留物,将其活性转换为过氧酶. 这种变化加强了血.
科学领域:
- 生物化学和酶学 生物化学和酶学
- 蛋白质工程和结构生物学
背景情况:
- 细胞染色体P450酶 (CYPs) 是关键的血红硫酸单氧酶,参与催化氧气插入有机分子.
- 在I螺旋中保存的酸-酒精对通常有助于大多数CYP中的二氧化物激活.
- 在这种CYP199A4对中的氨酸残留物转化为谷氨酸会诱导过氧酶活性.
研究的目的:
- 研究CYP199A4中T252E突变诱导过氧酶活性的机制.
- 为了确定T252E突变体中血红素的远端水体连接体是否可以被各种连接体所取代.
- 了解过氧化酶突变体中改变的催化活性的结构基础.
主要方法:
- 对CYP199A4的局部定向突变产生T252E突变.
- 结晶学以确定突变酶的结构.
- 紫外线可见吸收光谱学,原生质谱学和电子磁共振来评估连接体结合.
主要成果:
- X射线晶体结构揭示了T252E突变体中引入的谷氨酸侧链和heme的遥远水联体之间的相互作用.
- 这种相互作用防止了水联体被基质快速取代.
- 没有一个测试的大型疏水性或捐赠体可以取代远端水体,即使是那些已知可以结合野生类型酶中的铁的酶.
结论:
- 这种T252E突变显著增强了CYP199A4.4中铁区域水联体的相互作用.
- 这种增强的连接体相互作用阻止了基质进入血红素,有效地关闭了单氧化酶通路.
- 无法取代水联体,为突变酶中观察到的转向过氧酶活性提供了一种机械解释.
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