PMID:9074628

Citation	Löbau, S, Weber, J and Senior, AE (1997) Nucleotide occupancy of F1-ATPase catalytic sites under crystallization conditions. FEBS Lett. 404:15-8
Abstract	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.
Links	PubMed
Keywords	Adenosine Triphosphate/chemistry; Binding Sites/genetics; Catalysis; Crystallization; Escherichia coli/enzymology; Mutation; Oligodeoxyribonucleotides/chemistry; Proton-Translocating ATPases/chemistry; Proton-Translocating ATPases/genetics
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