IDENTIFYING THE CATALYTIC RESIDUE OF THE ATPASE REACTION OF DNA GYRASE

We propose a mechanism for the hydrolysis of ATP by the DNA gyrase B p rotein in which Glu42 acts as a general base and His38 has a role in a ligning and polarizing the glutamate residue. We have tested this mech anism by site-directed mutagenesis, converting Glu42 to Ala, Asp, and Gln, and His38 to Ala. In the presence of wild-type A protein, B prote ins bearing the mutations Ala42 and Gln42 show no detectable supercoil ing or ATPase activities, while Asp42 and Ala38 proteins have reduced activities. In the DNA cleavage and relaxation reactions of gyrase, wh ich do not require ATP hydrolysis, wild-type and mutant proteins have similar activities. When the 43-kDa N-terminal fragment of the gyrase B protein (which hydrolyzes ATP) contained the mutations Ala42 or Gln4 2, ATP was bound but not hydrolyzed, supporting the idea that Glu42 is involved in hydrolysis but not nucleotide binding.