Nucleotide occupancy of F1-ATPase catalytic sites under crystallization conditions.
Using site-directed tryptophan fluorescence we studied nucleotide occupancy of the catalytic sites of Escherichia coli F1-ATPase, under conditions used previously for crystallization and X-ray structure analysis of the bovine mitochondrial enzyme [Abrahams et al. (1994) Nature 370, 621-628]. We found that only two of the three catalytic sites were filled in the E. coli enzyme under these conditions (250 microM MgAMPPNP plus 5 microM MgADP), consistent with what was reported in the bovine F1 X-ray structure. However, subsequent addition of a physiological concentration of MgATP readily filled the third catalytic site. Therefore the enzyme form seen in the X-ray structure results from the fact that it is obtained under sub-saturating nucleotide conditions. The data show that the X-ray structure is compatible with a catalytic mechanism in which all three F1-ATPase catalytic sites must fill with MgATP to initiate steady-state hydrolysis [e.g. Weber and Senior (1996) Biochim. Biophys. Acta 1275, 101-104]. The data further demonstrate that the site-directed tryptophan fluorescence technique can provide valuable support for F1 crystallography studies.
Löbau
S
S
Department of Biochemistry and Biophysics, University of Rochester Medical Center, NY 14642, USA.
Weber
J
J
Senior
A E
AE
D000255:Adenosine Triphosphate / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases