PMID:10387006
| Citation |
Nadanaciva, S, Weber, J and Senior, AE (1999) The role of beta-Arg-182, an essential catalytic site residue in Escherichia coli F1-ATPase. Biochemistry 38:7670-7 |
|---|---|
| Abstract |
Beta-Arg-182 in Escherichia coli F1-ATPase (beta-Arg-189 in bovine mitochondrial F1) is a residue which lies close to catalytic site bound nucleotide (Abrahams et al. (1994) Nature 370, 621-628). Here we investigated the role of this residue by characterizing two mutants, betaR182Q and betaR182K. Oxidative phosphorylation and steady-state ATPase activity of purified F1 were severely impaired by both mutations. Catalytic site nucleotide-binding parameters were measured using the fluorescence quench of beta-Trp-331 that occurred upon nucleotide binding to purified F1 from betaR182Q/betaY331W and betaR182K/betaY331W double mutants. It was found that (a) beta-Arg-182 interacts with the gamma-phosphate of MgATP, particularly at catalytic sites 1 and 2, (b) beta-Arg-182 has no functional interaction with the beta-phosphate of MgADP or with the magnesium of the magnesium-nucleotide complex in the catalytic sites, and (c) beta-Arg-182 is directly involved in the stabilization of the catalytic transition state. In these features the role of beta-Arg-182 resembles that of another positively charged residue in the catalytic site, the conserved lysine of the Walker A motif, beta-Lys-155. A further role of beta-Arg-182 is suggested, namely involvement in conformational change at the catalytic site beta-alpha subunit interface that is required for multisite catalysis. |
| Links |
PubMed Online version:10.1021/bi990663x |
| Keywords |
Adenosine Diphosphate/chemistry; Adenosine Triphosphate/chemistry; Aluminum/chemistry; Amino Acid Substitution/genetics; Arginine/chemistry; Arginine/genetics; Binding Sites/genetics; Catalysis; Escherichia coli/enzymology; Escherichia coli/genetics; Fluorine/chemistry; Glutamine/chemistry; Glutamine/genetics; Lysine/chemistry; Lysine/genetics; Mutagenesis, Site-Directed; Proton-Translocating ATPases/chemistry; Proton-Translocating ATPases/genetics; Spectrometry, Fluorescence; Tryptophan/chemistry; Tryptophan/genetics |
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