The dnaB protein of Escherichia coli: mechanism of nucleotide binding, hydrolysis, and modulation by dnaC protein.
The mechanism of nucleotide binding and hydrolysis by dnaB protein and dnaB X dnaC protein complex has been studied by using fluorescent nucleotide analogues. Binding of trinitrophenyladenosine triphosphate (TNP-ATP) or the corresponding diphosphate (TNP-ADP) results in a blue shift of the emission maximum and a severalfold amplification of the fluorescence emission of the nucleotide analogues. Scatchard analysis of TNP-ATP binding indicates that TNP-ATP binds with a high affinity (Kd = 0.87 microM) and a 8.5-fold enhancement of fluorescence emission of the nucleotide. Only three molecules of TNP-ATP or TNP-ADP bind per hexamer of dnaB protein in contrast to six molecules of ATP or ADP binding to a dnaB hexamer. TNP-ATP and TNP-ADP are both competitive inhibitors of single-stranded (SS) DNA-dependent ATPase activity of dnaB protein. TNP-AMP neither binds to dnaB protein nor inhibits the ATPase activity. Formation of dnaB X dnaC complex by dnaC protein results in diminution of the TNP-ATP fluorescence enhancement and a concomitant decrease in the SS DNA-dependent ATPase activity. Kinetic analysis of the ATPase activity of dnaB X dnaC complex indicates that the decrease in the ATPase activity on complex formation is due to a reduction of the maximal velocity (Vmax). The dnaB protein hydrolyzes both TNP-ATP and dATP, however, with an extremely slow rate in the presence of single-stranded M13 DNA. The 2'-OH group of the nucleotide most likely plays an important role in the hydrolysis reaction but not in the nucleotide binding.
D000227:Adenine Nucleotides / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods
D000227:Adenine Nucleotides; D001426:Bacterial Proteins; D000251:Adenosine Triphosphatases