相关实验视频
Updated: May 20, 2026

07:38
Measuring In Vitro ATPase Activity for Enzymatic Characterization
Published on: August 23, 2016
乙醇pyruvylshikimate 3-酸盐 (EPSP) 合成酶 (AroA) 催化EPSP水解的过渡状态分析
Meiyan Lou1, Steven K Burger, Meghann E Gilpin
1Department of Chemistry & Chemical Biology, and †Department of Biochemistry & Biomedical Sciences, McMaster University , 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada.
Journal of the American Chemical Society
|July 7, 2012
概括
酶催化质子转移到碳是很困难的. 乙醇pyruvylshikimate 3-酸盐合成酶 (AroA) 使用早期的过渡状态来稳定关键的中间体,克服这一挑战.
科学领域:
- 生物化学 生物化学
- 酶催化酶的催化作用
- 化学动力学 化学动力学
背景情况:
- 由于高能障碍和不利的热力学,对碳原子的质子转移带来了重大的催化障碍.
- 乙醇pyruvylshikimate 3-酸盐合成酶 (EPSP合成酶,AroA) 面临的挑战是在它的反向反应中对醇pyruvate (EPSP) 的甲基碳进行质子化.
研究的目的:
- 分析AroA催化EPSP水解的过渡状态,作为酶逆反应的模拟物.
- 研究甲碳质子化的机制,并确定AroA的催化策略.
主要方法:
- 利用动态同位素效应 (KIEs),包括3-(14) C和溶剂KIEs,用于过渡状态分析.
- 开发了一种无机酸盐清理系统,以管理无处不在的酸盐度.
- 采用计算建模来分析过渡状态结构,并与实验数据进行比较.
主要成果:
- 反应通过不可逆转的C3质子化逐步进行,形成EPSP离子中间体 (AH(‡) *AN机制).
- 一个大型的实验3-(14) C KIE (1.032 ± 0.005) 表明C3与质子运动之间存在强烈的合.
- 观察到非常低的溶剂KIE (0.97±0.04),与早期过渡状态一致.
- 一个大型的2~14C KIE显示,通过Asp313和Glu341.1的"静电三明治"来稳定C2正电荷.
结论:
- AroA通过比酸催化反应更早地转移过渡状态来促进催化,从而实现显著的哈蒙德转移.
- 该酶的催化策略涉及稳定EPSP离子中间体的正电荷.
- 计算模型证实了实验结果,强调了活性部位残留物在催化中的作用.
相关概念视频
ATP Synthase: Mechanism
In animals, the mitochondrial F1F0 ATP synthase is the key protein that synthesizes ATP molecules through a complex catalytic mechanism. While the nuclear genome encodes the majority of ATP synthase subunits, the mitochondrial genome encodes some of the enzyme's most critical components. The formation of this multi-subunit enzyme is a complex multi-step process regulated at the level of transcription, translation, and assembly. Defects in one or more of these steps can result in decreased ATP...
Acid-Catalyzed Ring-Opening of Epoxides
Epoxides that are three-membered ring systems are more reactive than other cyclic and acyclic ethers. The high reactivity of epoxides originates from the strain present in the ring. This ring strain acts as a driving force for epoxides to undergo ring-opening reactions either with halogen acids or weak nucleophiles in the presence of mild acid. The acid catalyst converts the epoxide oxygen, a poor leaving group, into an oxonium ion, a better leaving group, making the reaction feasible. The...
ATP Energy Storage and Release
ATP is a highly unstable molecule. Unless quickly used to perform work, ATP spontaneously dissociates into ADP and inorganic phosphate (Pi), and the free energy released during this process is lost as heat. The energy released by ATP hydrolysis is used to perform work inside the cell and depends on a strategy called energy coupling. Cells couple the exergonic reaction of ATP hydrolysis with endergonic reactions, allowing them to proceed.
One example of energy coupling using ATP involves a...
One example of energy coupling using ATP involves a...
E2 Reaction: Kinetics and Mechanism
SN2 substitutions and E2 eliminations of alkyl halides proceed via a concerted pathway. While the nucleophile attacks the alpha carbon in SN2 reactions, it functions as a strong base and abstracts a beta hydrogen in the E2 mechanism. The rate-limiting transition state in E2 elimination reactions is characterized by partially broken carbon–hydrogen and carbon–halogen bonds and a partially formed pi bond between the alpha and beta carbons. The beta hydrogen and halide are eliminated...
ATP Synthase: Structure
ATP synthase or ATPase is among the most conserved proteins found in bacteria, mammals, and plants. This enzyme can catalyze a forward reaction in response to the electrochemical gradient, producing ATP from ADP and inorganic phosphate. ATP synthase can also work in a reverse direction by hydrolyzing ATP and generating an electrochemical gradient. Different forms of ATP synthases have evolved special features to meet the specific demands of the cell. Based on their specific feature, ATP...
Sharpless Epoxidation
The conversion of allylic alcohols into epoxides using the chiral catalyst was discovered by K. Barry Sharpless and is known as Sharpless epoxidation. The use of a chiral catalyst enables the formation of one enantiomer of the product in excess. This chiral catalyst is mainly a chiral complex of titanium tetraisopropoxide and tartrate ester (specific stereoisomer). The stereoisomer used in the chiral catalyst dictates the formation of the enantiomer of the product. In other words, the use of...

