X-ray studies of metalloenzyme regulation and catalysis

金属酶调节和催化的 X 射线研究

• 批准号: 6877080
• 负责人: CATHERINE L DRENNAN
• 金额: $ 31.5万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6877080
• 关键词: DNA replication; Escherichia coli; Lactobacillus; NAD(H) phosphate; NAD(P)H oxidoreductase; X ray crystallography; active sites; allosteric site; binding sites; catalyst; cobamide; crystallization; deoxyadenosines; dimer; enzyme activity; enzyme mechanism; enzyme structure; metalloenzyme; ribonucleotide reductase; site directed mutagenesis; thioredoxin; vitamin B12 coenzyme

The broad, long term goal of this research is to elucidate the role of conformational change in catalysis and enzyme regulation. Our research will emphasize the allosteric regulation and inter- and intramolecular communication of riboucleotide reductases (RNRs). These amazing enzymes utilize organic radical-based chemistry to convert nucleotides to deoxynucleotides. Allosteric regulation of RNR enzymes serves to maintain an appropriate supply of each of four deoxynucleotides (dNTPs) needed for DNA replication and repair. As a result of these cellular activities, RNRs are actively pursued as targets for antibacterial, antiviral, and antitumor therapies. Gemcitabine (2',2'-difluoro-2'-deoxycytidine), a mechanism-based inhibitor of RNRs, was recently approved by the FDA for treatment of pancreatic cancer and demonstrates the medical importance of these enzymes. The proposed research is intended to identify the conformational changes involved in RNR re-reduction by thioredoxin/thioredoxin reductase/NADPH, to identify the role of conformational change in the allosteric regulation of radical formation, and to identify the structural basis for allosteric regulation of enzyme specificity. These goals will be achieved by crystallographic analysis of the least complex members of the ribonucleotide reductase family, the B12-dependent class II enzymes. We will complement structural data with biochemical characterization and single crystal spectroscopic techniques.

这项研究的广泛、长期目标是阐明构象变化在催化和酶调节中的作用。 我们的研究将强调核糖核苷酸还原酶（RNR）的变构调节以及分子间和分子内通讯。 这些令人惊奇的酶利用有机自由基化学将核苷酸转化为脱氧核苷酸。 RNR 酶的变构调节有助于维持 DNA 复制和修复所需的四种脱氧核苷酸 (dNTP) 的适当供应。 由于这些细胞活性，RNR 作为抗菌、抗病毒和抗肿瘤治疗的靶点被积极研究。吉西他滨（2',2'-二氟-2'-脱氧胞苷）是一种基于机制的 RNR 抑制剂，最近被 FDA 批准用于治疗胰腺癌，并证明了这些酶的医学重要性。 拟议的研究旨在确定硫氧还蛋白/硫氧还蛋白还原酶/NADPH重新还原RNR所涉及的构象变化，确定构象变化在自由基形成变构调节中的作用，并确定酶变构调节的结构基础特异性。 这些目标将通过对核糖核苷酸还原酶家族中最简单的成员（B12 依赖性 II 类酶）进行晶体学分析来实现。 我们将通过生化表征和单晶光谱技术补充结构数据。