ATP synthase: Conditions under which all catalytic sites of the F1 moiety are kinetically equivalent in hydrolyzing ATP

Baltazar D. Reynafarje, Peter L Pedersen

Research output: Contribution to journalArticle

Abstract

Conditions have been reported under which the F1 moiety of bovine heart ATP synthase catalyzes the hydrolysis of ATP by an apparently cooperative mechanism in which the slow rate of hydrolysis at a single catalytic site (unisite catalysis) is enhanced more than 106-fold when ATP is added in excess to occupy one or both of the other two catalytic sites (multisite catalysis) (Cross, R. L., Grubmeyer, C., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12101-12105). In the novel studies reported here, and in contrast to the earlier report, we have (a) monitored the kinetics of ATP hydrolysis of F1 by using nucleotide-depleted preparations and a highly sensitive chemiluminescent assay; (b) followed the reaction immediately upon addition of F1 to ATP, rather than after prior incubation with ATP; and (c) used a reaction medium with Pi as the only buffer. The following observations were noted. First, regardless of the source of enzyme, bovine or rat, and catalytic conditions (unisite or multisite), the rates of hydrolysis depend on ATP concentration to the first power. Second, the first order rate constant for ATP hydrolysis remains relatively constant under both unisite and multisite conditions declining only slightly at high ATP concentration. Third, the initial rates of ATP hydrolysis exhibit Michaelis-Menten kinetic behavior with a single Vmax exceeding 100 μmol of ATP hydrolyzed per min/mg of F1 (turnover number = 635 s-1) and a single Km for ATP of about 57 μM. Finally, the reaction is inhibited markedly by low concentrations of ADP. It is concluded that, under the conditions described here, all catalytic sites that participate in the hydrolysis of ATP within the F1 moiety of mitochondrial ATP synthase function in a kinetically equivalent manner.

Original languageEnglish (US)
Pages (from-to)32546-32550
Number of pages5
JournalJournal of Biological Chemistry
Volume271
Issue number51
StatePublished - 1996

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Catalytic Domain
Adenosine Triphosphate
Hydrolysis
Catalysis
Mitochondrial Proton-Translocating ATPases
Luminescent Measurements
Kinetics
Adenosine Diphosphate
Rats
Rate constants
Assays
Buffers
Nucleotides

ASJC Scopus subject areas

  • Biochemistry

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ATP synthase : Conditions under which all catalytic sites of the F1 moiety are kinetically equivalent in hydrolyzing ATP. / Reynafarje, Baltazar D.; Pedersen, Peter L.

In: Journal of Biological Chemistry, Vol. 271, No. 51, 1996, p. 32546-32550.

Research output: Contribution to journalArticle

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