O'Dea M H, Tamura J K, Gellert M
Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0540, USA.
J Biol Chem. 1996 Apr 19;271(16):9723-9. doi: 10.1074/jbc.271.16.9723.
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 Lys103 and Lys110 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 103 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-beta,gamma-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 Lys110 retains all activities. However, the affinity of ATP is decreased. The DNA supercoiling activity of gyrase A2B2, 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.
我们之前报道过，ATP亲和类似物吡哆醛5'-二磷酸-5'-腺苷（PLP-AMP）可特异性标记大肠杆菌DNA促旋酶，这导致ATP依赖性反应受到抑制。已发现该类似物共价结合在促旋酶B亚基的Lys103和Lys110位点上（田村，J.K.，和盖勒特，M.（1990年）《生物化学杂志》265，21342 - 21349）。在本研究中，通过定点诱变来研究这两个赖氨酸残基的重要性。Lys 103的取代导致ATP依赖性功能丧失。这些突变体无法使DNA超螺旋、水解ATP或结合不可水解的ATP类似物5'-腺苷-β，γ-亚氨基二磷酸（ADPNP）。促旋酶的非ATP依赖性功能，如负超螺旋DNA的松弛和恶喹酸诱导的双链DNA切割，不受这些突变的影响，这表明突变的B亚基能与A亚基正确组装。Lys110被取代的促旋酶保留了所有活性。然而，ATP的亲和力降低。用不同比例的无活性突变体和野生型促旋酶B亚基重构的四聚体促旋酶A2B2的DNA超螺旋活性与一种DNA超螺旋机制一致，该机制要求两个B亚基在ATP结合和水解中相互依赖的活性。
