EcoRI DNA Methylase: Catalytic and Recognition Mechanisms

PI建议测试原核生物EcoRI DNA甲基转移酶(MTase)是否通过半胱氨酸223和腺嘌呤的C6位的共价中间体催化腺嘌呤N6位的甲基化。这将通过构建和测试含有丝氨酸、甘氨酸或丙氨酸的突变酶来测试，以取代半胱氨酸223。共价中间体的存在将通过半胱氨酸223和抑制剂之间形成共价键的机制抑制剂来进一步测试，该抑制剂旨在通过形成共价键来激活酶。除了阐明这种酶的化学机制外，这些结果还可能提供一类新的高度特异的抑制剂，并为设计具有新的底物特异性和作用机制的(生物)催化剂提供见解。使用随机和非随机的体外诱变方法，将提供该酶的底物专一性。特异性突变体将通过分子选择和利用聚合酶链式反应技术进行基因扩增来鉴定。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的DNA修饰机制，并可能有助于设计具有新的特异性的试剂。

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.