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相关概念视频

Ligand Binding and Linkage00:49

Ligand Binding and Linkage

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Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence...
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Enzymes02:34

Enzymes

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Inside living organisms, enzymes act as catalysts for many biochemical reactions involved in cellular metabolism. The role of enzymes is to reduce the activation energies of biochemical reactions by forming complexes with its substrates. The lowering of activation energies favor an increase in the rates of biochemical reactions.
Enzyme deficiencies can often translate into life-threatening diseases. For example, a genetic abnormality resulting in the deficiency of the enzyme G6PD...
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Induced-fit Model01:13

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Most chemical reactions in cells require enzymes—biological catalysts that speed up the reaction without being consumed or permanently changed. They reduce the activation energy needed to convert the reactants into products. Enzymes are proteins, that usually work by binding to a substrate—a reactant molecule that they act upon.
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Binding sites linkages can regulate a protein's function.  For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
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Groups of proteins may form a complex where each protein in this complex has a different role in the overall execution of the complex’s function. Often some of the proteins in the complex can be replaced by a closely related variant to give a complex that contains many of the same components yet is functionally distinct.
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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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相关实验视频

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Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
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两基质酶工程使用小型库,将两个不同的变异系的基质偏好结合起来.

Arka Mukhopadhyay1, Kersti Karu2, Paul A Dalby3

  • 1Department of Biochemical Engineering, UCL, Bernard Katz Building, Gower Street, London, WC1E 6BT, UK.

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概括

酶工程通过使新的基质组合成为可能,推进了生物催化. 研究人员成功地将突变结合起来,创建了具有显著改善活性的新型酶变体,用于特定的两基质反应.

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科学领域:

  • 生物催化和酶工程 生物催化和酶工程
  • 合成生物学 合成生物学
  • 蛋白质工程是一种蛋白质工程.

背景情况:

  • 酶工业化需要更广泛的基质范围和高的催化活性.
  • 由于相互连接的活性站点作用,为新型的两基质反应设计酶是复杂的.

研究的目的:

  • 开发一种结合有益突变的方法,以便为新的两基质反应重新编程酶活性位点.
  • 增强酶对替代基质的催化活性.

主要方法:

  • 设计了小型突变库,在关键突变部位结合了天然和非天然的氨基酸.
  • 研究的酶变体用于改善3 - 甲基酸 (3-FBA) 和pyruvate之间的反应的催化.
  • 利用计算对接来分析活性部位的结构变化和基质-酶相互作用.

主要成果:

  • 实现了高达630倍的催化速率 (kcat) 在3-FBA和pyruvate反应.
  • 确定了增强3-FBA与胺-胺-酸盐 (TPP) 中间体的近距离的特定突变.
  • 证明了酶活性位点的成功重编程,用于特定的两基质转换.

结论:

  • 小图书馆允许快速,插入和运行重新编程酶活性位点.
  • 这种方法有助于为各种两基质反应创建新型生物催化剂.
  • 通过扩大基质接受度和催化效率,为工业应用推进酶工程.