We have previously reported specific labeling of Escherichia coli DNA gyrase by the ATP affinity analog pyridoxal 5`-diphospho-5`adenosine (PLP-AMP), which resulted in inhibition of ATP-dependent reactions. The analog was found to be covalently bound at Lys and Lys on the gyrase B subunit (Tamura, J. K., and Gellert, M.(1990) J. Biol. Chem. 265, 21342-21349). In this study, the importance of these two lysine residues is examined by site-directed mutagenesis.

Substitutions of Lys result in the loss of ATP-dependent functions. These mutants are unable to supercoil DNA, to hydrolyze ATP, or to bind a nonhydrolysable ATP analog, 5`-adenylyl-,-imidodiphosphate (ADPNP). The ATP-independent functions of gyrase, such as relaxation of negatively supercoiled DNA and oxolinic acid-induced cleavage of double-stranded DNA, are unaffected by these mutations, suggesting that the mutant B subunits are assembling correctly with the A subunits. Gyrase with substitutions of Lys retains all activities. However, the affinity of ATP is decreased. The DNA supercoiling activity of gyrase AB tetramers reconstituted with varying ratios of inactive mutant and wild-type gyrase B subunits is consistent with a mechanism of DNA supercoiling that requires the interdependent activity of both B subunits in ATP binding and hydrolysis.

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