Transient kinetic studies reveal isomerization steps along the kinetic pathway of Thermus thermophilus 3-isopropylmalate dehydrogenase

Éva Gráczer, Corinne Lionne, P. Závodszky, Laurent Chaloin, M. Vas

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

To identify the rate-limiting step(s) of the 3-isopropylmalate dehydrogenase-catalysed reaction, time courses of NADH production were followed by stopped flow (SF) and quenched flow (QF). The steady state kcat and Km values did not vary between enzyme concentrations of 0.1 and 20 μm. A burst phase of NADH formation was shown by QF, indicating that the rate-limiting step occurs after the redox step. The kinetics of protein conformational change(s) induced by the complex of 3-isopropylmalate with Mg2+ were followed by using the fluorescence resonance energy transfer signal between protein tryptophan(s) and the bound NADH. A reaction scheme was proposed by incorporating the rate constant of a fast protein conformational change (possibly domain closure) derived from the separately recorded time-dependent formation of the fluorescence resonance energy transfer signal. The rate-limiting step seems to be another slower conformational change (domain opening) that allows product release. The time course of NADH production during the pre-steady state of 3-isopropylmalate dehydrogenase (IPMDH) catalysed reaction, followed by the fast kinetic method quenched flow (QF), is possibly limited by the occurrence of substrate-caused protein conformational change(s), e.g. domain closure, as reflected by formation of a FRET (fluorescence resonance energy transfer) signal between protein tryptophan(s) and the bound NADH.

Original languageEnglish
Pages (from-to)1764-1772
Number of pages9
JournalFEBS Journal
Volume280
Issue number8
DOIs
Publication statusPublished - Apr 2013

Fingerprint

3-Isopropylmalate Dehydrogenase
Thermus thermophilus
Isomerization
NAD
Fluorescence Resonance Energy Transfer
Kinetics
Proteins
Tryptophan
Oxidation-Reduction
Rate constants
Substrates
Enzymes

Keywords

  • 3-isopropylmalate dehydrogenase
  • fluorescence resonance energy transfer
  • hypochromic effect
  • reaction mechanism
  • transient kinetics

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Transient kinetic studies reveal isomerization steps along the kinetic pathway of Thermus thermophilus 3-isopropylmalate dehydrogenase. / Gráczer, Éva; Lionne, Corinne; Závodszky, P.; Chaloin, Laurent; Vas, M.

In: FEBS Journal, Vol. 280, No. 8, 04.2013, p. 1764-1772.

Research output: Contribution to journalArticle

@article{518f4d8cb5d54d7ca02f2dbe33b805ba,
title = "Transient kinetic studies reveal isomerization steps along the kinetic pathway of Thermus thermophilus 3-isopropylmalate dehydrogenase",
abstract = "To identify the rate-limiting step(s) of the 3-isopropylmalate dehydrogenase-catalysed reaction, time courses of NADH production were followed by stopped flow (SF) and quenched flow (QF). The steady state kcat and Km values did not vary between enzyme concentrations of 0.1 and 20 μm. A burst phase of NADH formation was shown by QF, indicating that the rate-limiting step occurs after the redox step. The kinetics of protein conformational change(s) induced by the complex of 3-isopropylmalate with Mg2+ were followed by using the fluorescence resonance energy transfer signal between protein tryptophan(s) and the bound NADH. A reaction scheme was proposed by incorporating the rate constant of a fast protein conformational change (possibly domain closure) derived from the separately recorded time-dependent formation of the fluorescence resonance energy transfer signal. The rate-limiting step seems to be another slower conformational change (domain opening) that allows product release. The time course of NADH production during the pre-steady state of 3-isopropylmalate dehydrogenase (IPMDH) catalysed reaction, followed by the fast kinetic method quenched flow (QF), is possibly limited by the occurrence of substrate-caused protein conformational change(s), e.g. domain closure, as reflected by formation of a FRET (fluorescence resonance energy transfer) signal between protein tryptophan(s) and the bound NADH.",
keywords = "3-isopropylmalate dehydrogenase, fluorescence resonance energy transfer, hypochromic effect, reaction mechanism, transient kinetics",
author = "{\'E}va Gr{\'a}czer and Corinne Lionne and P. Z{\'a}vodszky and Laurent Chaloin and M. Vas",
year = "2013",
month = "4",
doi = "10.1111/febs.12191",
language = "English",
volume = "280",
pages = "1764--1772",
journal = "FEBS Journal",
issn = "1742-464X",
publisher = "Wiley-Blackwell",
number = "8",

}

TY - JOUR

T1 - Transient kinetic studies reveal isomerization steps along the kinetic pathway of Thermus thermophilus 3-isopropylmalate dehydrogenase

AU - Gráczer, Éva

AU - Lionne, Corinne

AU - Závodszky, P.

AU - Chaloin, Laurent

AU - Vas, M.

PY - 2013/4

Y1 - 2013/4

N2 - To identify the rate-limiting step(s) of the 3-isopropylmalate dehydrogenase-catalysed reaction, time courses of NADH production were followed by stopped flow (SF) and quenched flow (QF). The steady state kcat and Km values did not vary between enzyme concentrations of 0.1 and 20 μm. A burst phase of NADH formation was shown by QF, indicating that the rate-limiting step occurs after the redox step. The kinetics of protein conformational change(s) induced by the complex of 3-isopropylmalate with Mg2+ were followed by using the fluorescence resonance energy transfer signal between protein tryptophan(s) and the bound NADH. A reaction scheme was proposed by incorporating the rate constant of a fast protein conformational change (possibly domain closure) derived from the separately recorded time-dependent formation of the fluorescence resonance energy transfer signal. The rate-limiting step seems to be another slower conformational change (domain opening) that allows product release. The time course of NADH production during the pre-steady state of 3-isopropylmalate dehydrogenase (IPMDH) catalysed reaction, followed by the fast kinetic method quenched flow (QF), is possibly limited by the occurrence of substrate-caused protein conformational change(s), e.g. domain closure, as reflected by formation of a FRET (fluorescence resonance energy transfer) signal between protein tryptophan(s) and the bound NADH.

AB - To identify the rate-limiting step(s) of the 3-isopropylmalate dehydrogenase-catalysed reaction, time courses of NADH production were followed by stopped flow (SF) and quenched flow (QF). The steady state kcat and Km values did not vary between enzyme concentrations of 0.1 and 20 μm. A burst phase of NADH formation was shown by QF, indicating that the rate-limiting step occurs after the redox step. The kinetics of protein conformational change(s) induced by the complex of 3-isopropylmalate with Mg2+ were followed by using the fluorescence resonance energy transfer signal between protein tryptophan(s) and the bound NADH. A reaction scheme was proposed by incorporating the rate constant of a fast protein conformational change (possibly domain closure) derived from the separately recorded time-dependent formation of the fluorescence resonance energy transfer signal. The rate-limiting step seems to be another slower conformational change (domain opening) that allows product release. The time course of NADH production during the pre-steady state of 3-isopropylmalate dehydrogenase (IPMDH) catalysed reaction, followed by the fast kinetic method quenched flow (QF), is possibly limited by the occurrence of substrate-caused protein conformational change(s), e.g. domain closure, as reflected by formation of a FRET (fluorescence resonance energy transfer) signal between protein tryptophan(s) and the bound NADH.

KW - 3-isopropylmalate dehydrogenase

KW - fluorescence resonance energy transfer

KW - hypochromic effect

KW - reaction mechanism

KW - transient kinetics

UR - http://www.scopus.com/inward/record.url?scp=84876296940&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84876296940&partnerID=8YFLogxK

U2 - 10.1111/febs.12191

DO - 10.1111/febs.12191

M3 - Article

VL - 280

SP - 1764

EP - 1772

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-464X

IS - 8

ER -