Enzyme Structure-Function Relationships

酶结构与功能的关系

• 批准号: 9728162
• 负责人: Stephen Remington
• 金额: $ 34.5万
• 依托单位: University of Oregon Eugene
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-03-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9728162&HistoricalAwards=false
• 关键词: Enzyme; Structure; Function; Relationships

9728162 Remington The primary focus of this investigation will be to determine three dimensional structures of enzymes that catalyze the deprotonation of a simple carbon acid, acetylCoenzyme A, as a first step in the overall reaction. The enzymes are allosteric (regulated) Azotobacter citrate synthase and Escherichia coli malate synthase, which are unrelated in amino acid sequence but catalyze virtually identical reactions. A wide variety of acetylCoenzyme A substrate analogs, that vary in their ability to mimic proposed reaction intermediates, are available or are straightforward to synthesize. The structures of complexes of these analogs with the two enzymes will be determined by high resolution X-ray crystallography, and the results will be compared to those already obtained for the same compounds bound to nonallosteric chicken citrate synthase, which have been determined at up to 1.5A resolution. In addition, some active-site mutants of chicken citrate synthase will be created and their structures determined in order to ascertain the functional role of certain amino acids. A minor focus of this project, the structure of yeast serine carboxypeptidase in complex with an extremely tight binding, new class of cytosolic proteinase inhibitor (25 kDa Ic, Ki = 10-10 M) will be determined. The complex has been crystallized and the structure will be determined by standard molecular replacement methods using the known structure of serine carboxypeptidase. The significance of the major focus of the project is that it is not understood how an enzyme can deprotonate a carbon atom without using unusual cofactors. This reaction does not take place in solution under physiological conditions in the absence of a catalyst. By "freezing" two unrelated enzymes that catalyze this reaction as the first step and comparing their structures, new insight will be generated as to the exact nature of the intermediate(s), leading to a more complete understanding of this crucial reaction. Compounds that are rela ted to the different proposed intermediates, but are unable to complete subsequent steps of the reaction, are available to make this study straightforward. The second approach will be to compare the active site structures of regulated citrate synthases with those of unregulated citrate synthases. Structural changes at the active site caused by the external effector (NADH) should yield additional clues as to how the intermediate is generated and thus how the reaction is catalyzed. Finally, mutant citrate synthases have already been generated that are severely impaired in one or more aspects of catalysis, and their structures, which have yet to be determined, are expected to be very informative. The significance of the minor focus of this project is that the serine carboxypeptidase inhibitor Ic has multiple roles in the cell as defined by genetic analysis, but is of unknown structure. Completion of the three dimensional structure of the complex of serine carboxypeptidase with the inhibitor will therefore reveal a novel structure, expanding the database of protein folds in general, and will additionally lead to a deeper understanding of the catalytic mechanism of serine carboxypeptidases, and how they recognize their unique substrates.

这项研究的主要重点将是确定催化简单碳酸乙酰辅酶a去质子化的酶的三维结构，作为整个反应的第一步。这两种酶分别是变构酶（受调节的）固氮菌柠檬酸合成酶和大肠杆菌苹果酸合成酶，它们的氨基酸序列不相关，但催化的反应几乎完全相同。各种各样的乙酰辅酶A底物类似物，其模拟所提出的反应中间体的能力各不相同，是可用的或直接合成的。这些类似物与这两种酶的配合物的结构将通过高分辨率x射线晶体学来确定，并将结果与已经获得的与非变构鸡柠檬酸合酶结合的相同化合物的结果进行比较，后者已在高达1.5A的分辨率下确定。此外，还将创建鸡柠檬酸合酶的一些活性位点突变体并确定其结构，以确定某些氨基酸的功能作用。本项目的一个次要重点是确定酵母丝氨酸羧肽酶与一种极紧密结合的复合物的结构，这是一类新的胞质蛋白酶抑制剂（25 kDa Ic, Ki = 10-10 M）。该配合物已结晶，其结构将通过使用已知丝氨酸羧肽酶结构的标准分子替代方法来确定。该项目的重要意义在于，目前还不清楚酶如何在不使用不寻常的辅因子的情况下使碳原子去质子化。在没有催化剂的生理条件下，这种反应不会在溶液中发生。通过“冷冻”催化该反应的两种不相关的酶作为第一步，并比较它们的结构，将对中间体的确切性质产生新的见解，从而更全面地了解这一关键反应。与所提出的不同中间体相关的化合物，但不能完成反应的后续步骤，可以使本研究变得简单。第二种方法是比较受调节的柠檬酸合酶和不受调节的柠檬酸合酶的活性位点结构。由外部效应物（NADH）引起的活性位点的结构变化应该为中间体如何产生以及如何催化反应提供额外的线索。最后，突变的柠檬酸合酶已经产生，在催化的一个或多个方面严重受损，其结构尚未确定，预计将提供非常有用的信息。本项目次要重点的意义在于，丝氨酸羧肽酶抑制剂Ic在细胞中具有遗传分析定义的多种作用，但结构未知。因此，完成丝氨酸羧肽酶与抑制剂复合物的三维结构将揭示一种新的结构，扩大蛋白质折叠数据库，并将进一步深入了解丝氨酸羧肽酶的催化机制，以及它们如何识别其独特的底物。