Y XAA 3K PROTEIN FAMILY--STRUCTURE AND MECHANISM

Y XAA 3K 蛋白家族——结构与机制

• 批准号: 2191816
• 负责人: JOHN M WHITELEY
• 金额: $ 17万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2191816
• 关键词: Leishmania; X ray crystallography; alcohol dehydrogenase; cofactor; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; high performance liquid chromatography; isomerase; lysine; polymerase chain reaction; protein folding; protein structure; site directed mutagenesis; spectrometry; tyrosine

Recent structural studies reveal that dihydropteridine reductase (DHPR) is a member of a large family of dinucleotide requiring enzymes comprising the short-chain dehydrogenases and carbohydrate epimerases. The known members of the differing groups have less than 20% sequence identity yet fold similarly. Each protein contains a glycine rich beta-alpha-beta-fold that can accommodate the AMP component of the dinucleotide cofactor and each contains a Y(Xaa)/3K motif in the vicinity of the active site. In fact, a total of at least five conserved amino acids occur throughout the family and appear essential to the creation of an environment in which a hydride transfer can occur. Three distinct reactions are catalyzed; reduction; oxidation, and epimerisation. In each case a polarized double bond such as C=N or C=O is created or reduced. Using specific site- directed mutagenesis and X-ray crystallography, this proposal intends to determine what structural features are necessary to allow such a group of sequentially disparate proteins to formulate apparently similar structures yet retain sufficient identity that three differing reactions can occur. A unique hypothesis common to each of the dinucleotide requiring enzyme mechanisms is that the tyrosine/lysine motif could be a novel method for facilitating proton exchange with the tyrosine phenolic group (pK about 10). Specific mutational and kinetic experiments that aim to confirm this concept are described for both DHPR and the 2,3-dihydro-2,3- dihydroxybenzoate dehydrogenase (DDBDH), which is to be purified, crystallized and structurally characterized. It is anticipated that the experimental results will demonstrate that structural folding patterns that ensure conserved amino acids retain a characteristic function are equally or more important than sequence when looking for protein behavioral patterns. As a corollary to the investigation it is also intended to derive the structure of the Leishmania ltdh encoding resistance protein, as it demonstrates unusual sequence similarities to the above family of enzymes.

最近的结构研究表明，二氢蝶啶还原酶（DHPR）是 需要二核苷酸的酶的大家族的成员，其包括 短链淀粉酶和碳水化合物差向异构酶。 已知 不同组的成员具有小于20%的序列同一性， 类似地折叠。 每种蛋白质都含有富含甘氨酸的β-α-β-折叠 其可以容纳二核苷酸辅因子的AMP组分， 每个在活性位点附近含有Y（Xaa）/3 K基序。 在 事实上，总共至少有五个保守的氨基酸存在于整个基因组中。 家庭和似乎至关重要的创造一个环境，其中一个 可以发生氢化物转移。 三种不同的反应被催化; 还原、氧化和差向异构化。 在每种情况下， 例如C=N或C=O的键被产生或还原。 使用特定网站- 定向诱变和X射线晶体学，该提案旨在 确定什么样的结构特征是必要的，以允许这样一组 顺序不同的蛋白质形成明显相似的结构 但仍保持足够的同一性，使得可以发生三种不同的反应。 一个独特的假设共同的每一个二核苷酸需要酶 酪氨酸/赖氨酸基序可能是一种新的方法， 促进与酪氨酸酚基的质子交换（pK约为 10）。 旨在证实这一点的特定突变和动力学实验 概念都描述了DHPR和2，3-二氢-2，3- 二羟基苯甲酸脱氢酶（DDBDH），其待纯化， 结晶和结构特征。 预计该 实验结果将表明，结构折叠模式， 确保保守氨基酸保留特征功能的方法， 在寻找蛋白质时， 行为模式 作为调查的一个必然结果，它还旨在得出 利什曼原虫ltdh编码抗性蛋白的结构，因为它 证明了与上述酶家族的不寻常的序列相似性。