EcoRI DNA Methylase: Catalytic and Recognition Mechanisms

EcoRI DNA 甲基化酶：催化和识别机制

• 批准号: 9018474
• 负责人: Norbert Reich
• 金额: $ 32.62万
• 依托单位: University of California-Santa Barbara
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-03-15 至 1995-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9018474&HistoricalAwards=false
• 关键词: EcoRI; DNA; Methylase; Catalytic; Recognition

The PI propose to test if the prokaryotic EcoRI DNA methyltransferase (MTase) catalyzes methylation at N6 of adenine via a covalent intermediate involving cysteine 223 and the C6 position of adenine. This will be tested by constructing and testing mutant enzymes containing serine, glycine or alanine in place of cysteine 223. The existence of a covalent intermediate will be further tested with a mechanism-based inhibitor designed to activate the enzyme by forming a covalent linkage between cysteine 223 and the inhibitor. In addition to elucidating the chemical mechanism of this enzyme, these results may provide a new class of highly specific inhibitors as well as providing insights into the design of (bio)catalysts with novel substrate specificities and mechanisms. using both random and nonrandom in vitro mutagenesis methods, the substrate specificity of the enzyme will be offered. Specificity mutants will be identified through molecular selection and gene amplification using polymerase chain reaction techniques. Understanding which amino acids are critical for DNA recognition will aid in understanding of sequence- specific DNA modification mechanisms and may aid the design of agents with novel specificities.

PI计划测试原核生物EcoRI DNA甲基转移酶（MTase）是否通过涉及半胱氨酸223和腺嘌呤C6位置的共价中间体催化腺嘌呤N6位点的甲基化。这将通过构建和测试含有丝氨酸、甘氨酸或丙氨酸代替半胱氨酸223的突变酶来验证。共价中间体的存在将通过一种基于机制的抑制剂进一步测试，该抑制剂设计通过在半胱氨酸223和抑制剂之间形成共价连接来激活酶。除了阐明该酶的化学机制外，这些结果还可能提供一类新的高特异性抑制剂，并为具有新底物特异性和机制的（生物）催化剂的设计提供见解。使用随机和非随机的体外诱变方法，酶的底物特异性将被提供。特异性突变体将通过分子选择和基因扩增使用聚合酶链反应技术进行鉴定。了解哪些氨基酸对DNA识别至关重要，将有助于理解序列特异性DNA修饰机制，并可能有助于设计具有新特异性的药物。