ATP-hydrolysis in chloroplasts

Uni-site catalysis and evidence for heterogeneity of catalytic sites

Petra Fromme, Peter Gräber

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

ATP-hydrolysis was measured with thylakoids under uni-site conditions. The rate constant for the ATP binding is 106 M-1 · s-1, the 'equilibrium constant' of the enzyme-substrate complex, EADPP1 EATP, is 0.4. The rate constants for the P1-release and the ADP-release are 0.23 s-1 and 0.07 s-1. This indicates that the enzyme carries out a complete turnover under uni-site conditions. The interaction of the nucleotide binding sites was investigated by first measuring the [γ-32P]ATP hydrolysis under uni-site conditions. After that, 1 mM unlabeled ATP was added, so that all ATP-binding sites were occupied. The [γ-32P]ATP, bound to the first site, was hydrolyzed at a rate of 0.8 ATP (CF0F1 s). In a second experiment, first unlabeled ATP was hydrolyzed under uni-site conditions, and then 1 mM [γ-32P]ATP was added. Under otherwise identical conditions, this allows the measurement of the rate of ATP hydrolysis catalyzed by the second (and possibly third) site. A rate of 40 ATP (CF0F1 s) was found. It was concluded that there exists a heterogeneity of the ATP-hydrolyzing sites on CF0F1 in thylakoid membranes.

Original languageEnglish (US)
Pages (from-to)187-194
Number of pages8
JournalBBA - Bioenergetics
Volume1020
Issue number2
DOIs
StatePublished - Nov 5 1990
Externally publishedYes

Fingerprint

Chloroplasts
Catalysis
Hydrolysis
Catalytic Domain
Adenosine Triphosphate
Thylakoids
Rate constants
Binding Sites
Equilibrium constants
Enzymes
Adenosine Diphosphate
Nucleotides
Membranes

Keywords

  • ATPase, H-
  • CFF
  • Chloroplast
  • Nucleotide binding site
  • Uni-site catalysis

ASJC Scopus subject areas

  • Biophysics

Cite this

ATP-hydrolysis in chloroplasts : Uni-site catalysis and evidence for heterogeneity of catalytic sites. / Fromme, Petra; Gräber, Peter.

In: BBA - Bioenergetics, Vol. 1020, No. 2, 05.11.1990, p. 187-194.

Research output: Contribution to journalArticle

@article{2eccb395467b40f9ac2dc1b5382b01b6,
title = "ATP-hydrolysis in chloroplasts: Uni-site catalysis and evidence for heterogeneity of catalytic sites",
abstract = "ATP-hydrolysis was measured with thylakoids under uni-site conditions. The rate constant for the ATP binding is 106 M-1 · s-1, the 'equilibrium constant' of the enzyme-substrate complex, EADPP1 EATP, is 0.4. The rate constants for the P1-release and the ADP-release are 0.23 s-1 and 0.07 s-1. This indicates that the enzyme carries out a complete turnover under uni-site conditions. The interaction of the nucleotide binding sites was investigated by first measuring the [γ-32P]ATP hydrolysis under uni-site conditions. After that, 1 mM unlabeled ATP was added, so that all ATP-binding sites were occupied. The [γ-32P]ATP, bound to the first site, was hydrolyzed at a rate of 0.8 ATP (CF0F1 s). In a second experiment, first unlabeled ATP was hydrolyzed under uni-site conditions, and then 1 mM [γ-32P]ATP was added. Under otherwise identical conditions, this allows the measurement of the rate of ATP hydrolysis catalyzed by the second (and possibly third) site. A rate of 40 ATP (CF0F1 s) was found. It was concluded that there exists a heterogeneity of the ATP-hydrolyzing sites on CF0F1 in thylakoid membranes.",
keywords = "ATPase, H-, CFF, Chloroplast, Nucleotide binding site, Uni-site catalysis",
author = "Petra Fromme and Peter Gr{\"a}ber",
year = "1990",
month = "11",
day = "5",
doi = "10.1016/0005-2728(90)90050-E",
language = "English (US)",
volume = "1020",
pages = "187--194",
journal = "Biochimica et Biophysica Acta - Bioenergetics",
issn = "0005-2728",
publisher = "Elsevier",
number = "2",

}

TY - JOUR

T1 - ATP-hydrolysis in chloroplasts

T2 - Uni-site catalysis and evidence for heterogeneity of catalytic sites

AU - Fromme, Petra

AU - Gräber, Peter

PY - 1990/11/5

Y1 - 1990/11/5

N2 - ATP-hydrolysis was measured with thylakoids under uni-site conditions. The rate constant for the ATP binding is 106 M-1 · s-1, the 'equilibrium constant' of the enzyme-substrate complex, EADPP1 EATP, is 0.4. The rate constants for the P1-release and the ADP-release are 0.23 s-1 and 0.07 s-1. This indicates that the enzyme carries out a complete turnover under uni-site conditions. The interaction of the nucleotide binding sites was investigated by first measuring the [γ-32P]ATP hydrolysis under uni-site conditions. After that, 1 mM unlabeled ATP was added, so that all ATP-binding sites were occupied. The [γ-32P]ATP, bound to the first site, was hydrolyzed at a rate of 0.8 ATP (CF0F1 s). In a second experiment, first unlabeled ATP was hydrolyzed under uni-site conditions, and then 1 mM [γ-32P]ATP was added. Under otherwise identical conditions, this allows the measurement of the rate of ATP hydrolysis catalyzed by the second (and possibly third) site. A rate of 40 ATP (CF0F1 s) was found. It was concluded that there exists a heterogeneity of the ATP-hydrolyzing sites on CF0F1 in thylakoid membranes.

AB - ATP-hydrolysis was measured with thylakoids under uni-site conditions. The rate constant for the ATP binding is 106 M-1 · s-1, the 'equilibrium constant' of the enzyme-substrate complex, EADPP1 EATP, is 0.4. The rate constants for the P1-release and the ADP-release are 0.23 s-1 and 0.07 s-1. This indicates that the enzyme carries out a complete turnover under uni-site conditions. The interaction of the nucleotide binding sites was investigated by first measuring the [γ-32P]ATP hydrolysis under uni-site conditions. After that, 1 mM unlabeled ATP was added, so that all ATP-binding sites were occupied. The [γ-32P]ATP, bound to the first site, was hydrolyzed at a rate of 0.8 ATP (CF0F1 s). In a second experiment, first unlabeled ATP was hydrolyzed under uni-site conditions, and then 1 mM [γ-32P]ATP was added. Under otherwise identical conditions, this allows the measurement of the rate of ATP hydrolysis catalyzed by the second (and possibly third) site. A rate of 40 ATP (CF0F1 s) was found. It was concluded that there exists a heterogeneity of the ATP-hydrolyzing sites on CF0F1 in thylakoid membranes.

KW - ATPase, H-

KW - CFF

KW - Chloroplast

KW - Nucleotide binding site

KW - Uni-site catalysis

UR - http://www.scopus.com/inward/record.url?scp=0025165464&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025165464&partnerID=8YFLogxK

U2 - 10.1016/0005-2728(90)90050-E

DO - 10.1016/0005-2728(90)90050-E

M3 - Article

VL - 1020

SP - 187

EP - 194

JO - Biochimica et Biophysica Acta - Bioenergetics

JF - Biochimica et Biophysica Acta - Bioenergetics

SN - 0005-2728

IS - 2

ER -