双碳酸盐作为质子捐赠体,用于含有Zn (II) 和Co (II) 的碳酸无水酶的催化
1Departments of Pharmacology and Biochemistry, University of Florida College of Medicine, Gainesville, FL 32610-0267, USA.
Journal of the American Chemical Society
|June 21, 2001
概括
这项研究揭示了二碳酸盐如何促进二氧化碳水酶II中的质子转移,二氧化碳水的关键酶. 这些发现通过分析氧同位素交换率来阐明催化机制.
科学领域:
- 生物化学 生物化学
- 酶动力学 酶动力学
- 结构生物学 结构生物学
背景情况:
- 人类二氧化碳无水酶II (HCA II) 是一种关键的酶,催化二氧化碳的可逆化.
- 了解精确的催化机制,包括质子转移步骤,对于酶工程和药物开发至关重要.
- 在HCA II中金属的替代提供了对活性部位残留物和金属离子在催化中的作用的见解.
研究的目的:
- 研究HCA II的催化循环中的速度限制步骤,特别是从活性部位释放的水.
- 阐明从二碳酸盐向活性位点金属结合氧化物的质子转移在氧气交换反应中的作用.
- 为了比较Co (II) 和Zn (II) 替代HCA II突变体和相关的古人类酶的催化机制.
主要方法:
- 在HCA II的局部定向突变发生,以阿拉宁取代His64,破坏本地质子穿.
- 使用质谱学分析二氧化碳和水之间的18O交换,这种交换由由Co (II) 和Zn (II) 替代的HCA II突变体催化.
- 对二碳酸盐依赖的氧气交换率和溶剂同位素效应的动态分析.
主要成果:
- 发现从Co(II) -HCA II突变的活性部位释放H2(18) O的速度取决于对金属结合氧化物的质子转移.
- 碳酸盐度的增加导致H2O释放率的和性增加,达到最多4 x 10 5 s 1 .
- 二甲II突变体的释放速度是H2O释放速度的10倍,但具有相似的CO2/HCO3-) 相互转换率,这表明碳酸直接将质子转移到结合的氧化物中.
结论:
- 双碳酸盐作为HCA II的活性位点中的金属结合氧化物的质子捐赠体.
- 由二碳酸盐进行质子捐赠的催化机制在人类和考古碳酸无水化合物之间保持着.
- 该研究提供了对质子转移途径和金属离子在碳酸无水酶催化中的作用的关键见解.
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