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，二氢吡啶还原酶（DHPR）是 大型二核苷酸家族的成员需要酶 短链脱氢酶和碳水化合物分配酶。 已知 不同群体的成员的序列身份小于20％ 类似地折叠。 每种蛋白质都有富含甘氨酸的β-alpha-beta蛋白 可以容纳二核苷酸辅因子的AMP组件，并且 每个都包含活性位点附近的Y（XAA）/3K基序。 在 事实，在整个过程中，总共至少五个保守的氨基酸发生 家庭，对于创造一个环境至关重要的环境至关重要 可以发生氢化物转移。 催化了三种不同的反应； 减少;氧化和表达。 在每种情况下都是两极分化的 创建或减少了诸如C = N或C = O之类的键。 使用特定站点 - 该提案的定向诱变和X射线晶体学旨在 确定必须进行哪些结构特征以允许这样的一组 依次分散的蛋白质以制定明显相似的结构 然而，保留了足够的身份，可能会发生三种不同的反应。 每种二核苷酸都需要酶的独特假设 机制是酪氨酸/赖氨酸基序可能是一种新颖的方法 促进与酪氨酸酚类群的质子交换（PK 10）。 旨在确认这一点的特定突变和动力学实验 DHPR和2,3-二氢-2,3-均描述了概念 二羟基苯甲酸酯脱氢酶（DDBDH），应纯化， 结晶和结构表征。 预计 实验结果将证明结构折叠模式 确保保守的氨基酸保留特征功能是 寻找蛋白质时的序列比序列更重要 行为模式。 作为调查的推论，它也旨在得出 Leishmania LTDH编码抗性蛋白的结构 证明了与上述酶家族的异常序列相似性。