EcoRI DNA Methylase: Catalytic and Recognition Mechanisms

PI建议测试原核EcoRI DNA是否 甲基转移酶（MTase）催化N6的甲基化， 腺嘌呤通过共价中间体涉及半胱氨酸223 和腺嘌呤的C6位置。 这将由以下人员进行测试： 构建并测试含丝氨酸的突变酶， 甘氨酸或丙氨酸代替半胱氨酸223。 存在 共价中间体的量将进一步用 基于机制的抑制剂，旨在通过以下方式激活酶： 在半胱氨酸223和半胱氨酸223之间形成共价键， 抑制剂. 除了阐明化学机制外 这些结果可能会提供一种新的酶， 高度特异性的抑制剂，以及提供洞察， 新型基质（生物）催化剂的设计 特性和机制。 使用随机和 非随机体外诱变方法，底物 将提供酶的特异性。 特异性 将通过分子选择鉴定突变体， 聚合酶链反应基因扩增 技术.了解哪些氨基酸对 DNA识别将有助于理解序列- 特定的DNA修饰机制，并可能有助于设计 具有新特异性的药物

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.