In vitro coupling of ATP hydrolysis to proteolysis in the Escherichia coli protease Lon forms mutant in the ATPase site

In order to identify amino acid residues involved in ATP hydrolysis by Escherichia Lon protease Lon or participating in the signal transduction from the ATPase domain to the proteolytic one, potentially important residues of the ATPase domain were substituted using site-directed mutagenesis, and the properties of the resulting mutant enzymes were studied. It was found that residues K362, T363 (Walker's motif A), and D423 (motif B) are involved in the catalysis of ATP hydrolysis. K362 and T363 also participate in the system of domain-domain coupling, whereas D423 does not play a significant role in this process. Residue D387 is important for ATPase activity; however, it is not a catalytically active residue, as was earlier postulated in the literature. Residue Y493 is also involved in the signal transduction from the ATPase domain to the proteolytic one.