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Titolo:
THE TRAPPING OF DIFFERENT CONFORMATIONS OF THE ESCHERICHIA-COLI F1 ATPASE BY DISULFIDE BOND FORMATION - EFFECT OF NUCLEOTIDE-BINDING AFFINITIES OF THE CATALYTIC SITES
Autore:
GRUBER G; CAPALDI RA;
Indirizzi:
UNIV OREGON,INST MOL BIOL EUGENE OR 97403 UNIV OREGON,INST MOL BIOL EUGENE OR 97403
Titolo Testata:
The Journal of biological chemistry
fascicolo: 51, volume: 271, anno: 1996,
pagine: 32623 - 32628
SICI:
0021-9258(1996)271:51<32623:TTODCO>2.0.ZU;2-2
Fonte:
ISI
Lingua:
ENG
Soggetto:
F1 ADENOSINE-TRIPHOSPHATASE; EPSILON-SUBUNIT; NONCATALYTIC SITES; OXIDATIVE-PHOSPHORYLATION; CRYOELECTRON MICROSCOPY; GAMMA-SUBUNIT; BETA-SUBUNIT; DIRECT PROBE; F1-ATPASE; FLUORESCENCE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
32
Recensione:
Indirizzi per estratti:
Citazione:
G. Gruber e R.A. Capaldi, "THE TRAPPING OF DIFFERENT CONFORMATIONS OF THE ESCHERICHIA-COLI F1 ATPASE BY DISULFIDE BOND FORMATION - EFFECT OF NUCLEOTIDE-BINDING AFFINITIES OF THE CATALYTIC SITES", The Journal of biological chemistry, 271(51), 1996, pp. 32623-32628

Abstract

Two mutants of the Escherichia coli F-1 ATPase, beta Y331W:E381C/epsilon S108C and alpha S411C/beta Y331W/epsilon S108C, have been used to relate nucleotide binding in catalytic sites with different interactions of the stalk-forming subunits gamma and epsilon at the alpha(3) beta(3) subunit domain, Essentially full yield cross-linking between beta+ gamma and beta + epsilon, or between alpha + gamma and alpha + epsilon was obtained in these mutants by Cu2+-induced disulfide bond formation, thereby trapping the enzyme in states with the small subunits interacting either with beta or alpha subunits, The presence of the Trp for beta Tyr-331 in both mutants allowed direct measurement of nucleotide occupancy of catalytic sites. Before cross-linking, Mg2+-ATP couldbe bound in all three catalytic sites in both mutants with a K-d of around 0.1 mu M for the highest affinity site and K-d values of approximately 2 mu M and 30-40 mu M for the second and third sites, respectively, In the absence of Mg2+, ATP also bound in all three catalytic sites but with a single low affinity (above 100 mu M) in both mutants, Cu2+-induced cross-linking of ECF(1) from the mutant beta Y331W:E381C/epsilon S108C had very little effect on nucleotide binding, The binding affinities of the three catalytic sites for Mg2+ ATP were not significantly altered from those obtained before cross-linking, and the enzymestill switched between cooperative binding and equal binding affinities of the three catalytic sites (when Mg2+ was absent). When the gammaand epsilon subunits were cross-linked to alpha subunits, ATP bindingin the highest affinity catalytic site was dramatically altered, Thissite became closed so that nucleotide (ATP or ADP) that had been bound into it prior to cross-linking was trapped and could not exchange out. Also, ATP or ADP could not enter this site, although empty, once the enzyme had been cross-linked, Finally, cross-linking of the gamma and epsilon to the alpha subunits prevented the switching between cooperative binding and the state where the three catalytic sites are equivalent, We argue that the conformation of the enzyme in which the small subunits are at alpha subunits occurs during functioning of the enzymein the course of the rotation of gamma and epsilon subunits within the alpha(3) beta(3) hexamer and that this may be the activated state for ATP synthesis.

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Documento generato il 13/08/20 alle ore 14:35:08