定位基在固醇异构酶中的催化贡献的评估
Vandana Lamba1, Filip Yabukarski1, Margaux Pinney1
1Department of Biochemistry, ‡Department of Chemistry, #Department of Chemical Engineering, §Stanford ChEM-H, Stanford University , Stanford, California 94305, United States.
Journal of the American Chemical Society
|July 14, 2016
概括
通过研究酶催化,本研究量化了基因在固醇异构酶 (KSI) 中的贡献. 使用外源性救援和多种碳酸基,它揭示了重要的催化作用,表明了除了定位之外的其他机制.
科学领域:
- 生物化学
- 酶动力学
- 蛋白质工程
背景情况:
- 在使用一般酸和的酶活性位点中,质子转移反应至关重要.
- 鉴定和量化这些残留物的催化贡献是具有挑战性的.
- 使用外源剂的有效度测量可以估计催化作用,但容易产生硬质和结合偏差.
研究的目的:
- 通过外源性救援来研究类异构酶 (KSI) 中一般基的催化作用.
- 在有效的度测量中评估和最大限度地减少度和定位效应.
- 确定一般基的催化作用在 *Comamonas testosteroni* KSI (tKSI) 和 *Pseudomonas putida* KSI (pKSI) 中.
主要方法:
- 包括一般基和附近残留物在内的KSI的系统性突变发生.
- 使用不同大小但相似的pKa的一系列碳酸基的外源性救援实验.
- 一个tKSI突变和双突变周期分析的X射线晶体学.
主要成果:
- 在tKSI中获得约5×10^4M和pKSI中获得10^3M的共识有效度.
- 结构分析显示一般基因突变没有显著的重组.
- 双重突变周期表明没有长期突变效应,验证了实验方法.
结论:
- 高的有效率强烈地表明了基因组的总基础的重大催化作用.
- 虽然定位可能起作用,但效应的大小意味着涉及到额外的催化机制.
- 外源性剂的广泛变化是必要的,以准确评估酶催化贡献.
相关概念视频
01:07Alkylation of β-Ketoester Enolates: Acetoacetic Ester Synthesis
4.8K
Acetoacetic ester synthesis is a method to obtain ketones from alkyl halides and β-keto esters. The reaction occurs in the presence of an alkoxide base that abstracts the acidic proton of the β-keto esters. The step results in an enolate ion which is doubly stabilized. The enolate then reacts with an alkyl halide via the SN2 process to produce an alkylated ester intermediate with a new C–C bond. The hydrolysis of the intermediate, followed by acidification, results in an...
4.8K
01:14Keto–Enol Tautomerism: Mechanism
8.2K
The keto and enol forms are known as tautomers and they constantly interconvert (or tautomerize) between the two forms under acid or base catalyzed conditions. Both the reactions involve the same steps—protonation and deprotonation— although in the reverse order.
8.2K
01:27Aldol Condensation with β-Diesters: Knoevenagel Condensation
4.0K
The Knoevenagel condensation is an aldol-type reaction involving the condensation of aldehydes or ketones with active methylene compounds such as β-diesters to produce substituted olefins.
4.0K
01:10Factors Affecting α-Alkylation of Ketones: Choice of Base
3.7K
α-Alkylation of ketones is achieved in the presence of alkyl halides and a base. The reaction proceeds via the formation of an enolate ion followed by nucleophilic substitution. The choice of base employed is essential as it is the key factor in determining the reaction outcome.
The reaction involving bases like EtO− whose conjugate acid EtOH (pKa = 15.9) is stronger than the ketone (pKa = 19.2) results in an equilibrium mixture with higher ketone concentration. As a consequence,...
The reaction involving bases like EtO− whose conjugate acid EtOH (pKa = 15.9) is stronger than the ketone (pKa = 19.2) results in an equilibrium mixture with higher ketone concentration. As a consequence,...
3.7K
01:22Reactions of Aldehydes and Ketones: Baeyer–Villiger Oxidation
5.2K
Baeyer–Villiger oxidation converts aldehydes to carboxylic acids and ketones to esters. The reaction uses peroxy acids or peracids and is often catalyzed by acid. The reaction is named after its pioneers, Adolf von Baeyer and Victor Villiger. The reaction is achieved by a wide range of peracids such as m-chloroperoxybenzoic acid (mCPBA), perbenzoic acid (C6H5COOOH), peracetic acid (CH3COOOH), hydrogen peroxide (H2O2), and tert-butyl hydroperoxide (t-BuOOH).
The carbonyl center is activated by...
The carbonyl center is activated by...
5.2K
01:33Regioselective Formation of Enolates
3.6K
As depicted in the figure below, the unsymmetrical ketones can form two possible enolates: less substituted or more substituted enolates. Usually, the thermodynamic enolates are formed from the more substituted α-carbon atom, while the kinetic enolates are formed faster by deprotonation from the less substituted position. The thermodynamic enolates have lower energy, so they are more stable. But the energy required to form kinetic enolates is less.
3.6K