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.
