Catalytic activity of the alpha3beta3gamma complex of F1-ATPase without noncatalytic nucleotide binding site.

TitleCatalytic activity of the alpha3beta3gamma complex of F1-ATPase without noncatalytic nucleotide binding site.
Publication TypeJournal Article
Year of Publication1997
AuthorsMatsui, T, Muneyuki, E, Honda, M, Allison, WS, Dou, C, Yoshida, M
JournalJ Biol Chem
Volume272
Issue13
Pagination8215-21
Date Published1997 Mar 28
ISSN0021-9258
KeywordsAdenosine Diphosphate, Adenosine Triphosphate, Bacillus, Binding Sites, Dimethylamines, Fluorescent Dyes, Kinetics, Peptide Mapping, Protein Conformation, Proton-Translocating ATPases, Structure-Activity Relationship, Surface-Active Agents
Abstract

A mutant alpha3beta3gamma complex of F1-ATPase from thermophilic Bacillus PS3 was generated in which noncatalytic nucleotide binding sites lost their ability to bind nucleotides. It hydrolyzed ATP at an initial rate with cooperative kinetics (Km(1), 4 microM; Km(2), 135 microM) similar to the wild-type complex. However, the initial rate decayed rapidly to an inactivated form. Since the inactivated mutant complex contained 1.5 mol of ADP/mol of complex, this inactivation seemed to be caused by entrapping inhibitory MgADP in a catalytic site. Indeed, the mutant complex was nearly completely inactivated by a 10 min prior incubation with equimolar MgADP. Analysis of the progress of inactivation after initiation of ATP hydrolysis as a function of ATP concentration indicated that the inactivation was optimal at ATP concentrations in the range of Km(1). In the presence of ATP, the wild-type complex dissociated the inhibitory [3H]ADP preloaded onto a catalytic site whereas the mutant complex did not. Lauryl dimethylamineoxide promoted release of preloaded inhibitory [3H]ADP in an ATP-dependent manner and partly restored the activity of the inactivated mutant complex. Addition of ATP promoted single-site hydrolysis of 2',3'-O-(2,4,6-trinitrophenyl)-ATP preloaded at a single catalytic site of the mutant complex. These results indicate that intact noncatalytic sites are essential for continuous catalytic turnover of the F1-ATPase but are not essential for catalytic cooperativity of F1-ATPase observed at ATP concentrations below approximately 300 microM.

DOI10.1074/jbc.272.13.8215
Alternate JournalJ. Biol. Chem.
Citation Key10.1074/jbc.272.13.8215
PubMed ID9079639