A link between hinge-bending domain motions and the temperature dependence of catalysis in 3-isopropylmalate dehydrogenase

István Hajdú, András Szilágyi, J. Kardos, P. Závodszky

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Enzyme function depends on specific conformational motions. We show that the temperature dependence of enzyme kinetic parameters can provide insight into these functionally relevant motions. While investigating the catalytic properties of IPMDH from Escherichia coli, we found that its catalytic efficiency (kcat/KM,IPM) for the substrate IPM has an unusual temperature dependence, showing a local minimum at ∼35°C. In search of an explanation, we measured the individual constants kcat and KM,IPM as a function of temperature, and found that the van 't Hoff plot of KM,IPM shows sigmoid-like transition in the 20-40°C temperature range. By means of various measurements including hydrogen-deuterium exchange and fluorescence resonance energy transfer, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperatures >30°C. The thermodynamic parameters of ligand binding determined by isothermal titration calorimetry as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. Because the binding of IPM is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with IPM binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency.

Original languageEnglish
Pages (from-to)5003-5012
Number of pages10
JournalBiophysical Journal
Volume96
Issue number12
DOIs
Publication statusPublished - 2009

Fingerprint

3-Isopropylmalate Dehydrogenase
Catalysis
Temperature
Deuterium Exchange Measurement
Enzymes
Fluorescence Resonance Energy Transfer
Calorimetry
Sigmoid Colon
Thermodynamics

ASJC Scopus subject areas

  • Biophysics

Cite this

A link between hinge-bending domain motions and the temperature dependence of catalysis in 3-isopropylmalate dehydrogenase. / Hajdú, István; Szilágyi, András; Kardos, J.; Závodszky, P.

In: Biophysical Journal, Vol. 96, No. 12, 2009, p. 5003-5012.

Research output: Contribution to journalArticle

@article{893afbaaa152414d8c60cfab74730603,
title = "A link between hinge-bending domain motions and the temperature dependence of catalysis in 3-isopropylmalate dehydrogenase",
abstract = "Enzyme function depends on specific conformational motions. We show that the temperature dependence of enzyme kinetic parameters can provide insight into these functionally relevant motions. While investigating the catalytic properties of IPMDH from Escherichia coli, we found that its catalytic efficiency (kcat/KM,IPM) for the substrate IPM has an unusual temperature dependence, showing a local minimum at ∼35°C. In search of an explanation, we measured the individual constants kcat and KM,IPM as a function of temperature, and found that the van 't Hoff plot of KM,IPM shows sigmoid-like transition in the 20-40°C temperature range. By means of various measurements including hydrogen-deuterium exchange and fluorescence resonance energy transfer, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperatures >30°C. The thermodynamic parameters of ligand binding determined by isothermal titration calorimetry as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. Because the binding of IPM is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with IPM binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency.",
author = "Istv{\'a}n Hajd{\'u} and Andr{\'a}s Szil{\'a}gyi and J. Kardos and P. Z{\'a}vodszky",
year = "2009",
doi = "10.1016/j.bpj.2009.04.014",
language = "English",
volume = "96",
pages = "5003--5012",
journal = "Biophysical Journal",
issn = "0006-3495",
publisher = "Biophysical Society",
number = "12",

}

TY - JOUR

T1 - A link between hinge-bending domain motions and the temperature dependence of catalysis in 3-isopropylmalate dehydrogenase

AU - Hajdú, István

AU - Szilágyi, András

AU - Kardos, J.

AU - Závodszky, P.

PY - 2009

Y1 - 2009

N2 - Enzyme function depends on specific conformational motions. We show that the temperature dependence of enzyme kinetic parameters can provide insight into these functionally relevant motions. While investigating the catalytic properties of IPMDH from Escherichia coli, we found that its catalytic efficiency (kcat/KM,IPM) for the substrate IPM has an unusual temperature dependence, showing a local minimum at ∼35°C. In search of an explanation, we measured the individual constants kcat and KM,IPM as a function of temperature, and found that the van 't Hoff plot of KM,IPM shows sigmoid-like transition in the 20-40°C temperature range. By means of various measurements including hydrogen-deuterium exchange and fluorescence resonance energy transfer, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperatures >30°C. The thermodynamic parameters of ligand binding determined by isothermal titration calorimetry as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. Because the binding of IPM is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with IPM binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency.

AB - Enzyme function depends on specific conformational motions. We show that the temperature dependence of enzyme kinetic parameters can provide insight into these functionally relevant motions. While investigating the catalytic properties of IPMDH from Escherichia coli, we found that its catalytic efficiency (kcat/KM,IPM) for the substrate IPM has an unusual temperature dependence, showing a local minimum at ∼35°C. In search of an explanation, we measured the individual constants kcat and KM,IPM as a function of temperature, and found that the van 't Hoff plot of KM,IPM shows sigmoid-like transition in the 20-40°C temperature range. By means of various measurements including hydrogen-deuterium exchange and fluorescence resonance energy transfer, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperatures >30°C. The thermodynamic parameters of ligand binding determined by isothermal titration calorimetry as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. Because the binding of IPM is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with IPM binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency.

UR - http://www.scopus.com/inward/record.url?scp=68949144754&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=68949144754&partnerID=8YFLogxK

U2 - 10.1016/j.bpj.2009.04.014

DO - 10.1016/j.bpj.2009.04.014

M3 - Article

VL - 96

SP - 5003

EP - 5012

JO - Biophysical Journal

JF - Biophysical Journal

SN - 0006-3495

IS - 12

ER -