Interactions of the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

Hyo Il Jung, Alan Cooper, Richard N. Perham

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

The enzymes pyruvate decarboxylase (E1) and dihydrolipoyl dehydrogenase (E3) bind tightly but in a mutually exclusive manner to the peripheral subunit-binding domain (PSBD) of dihydrolipoyl acetyltransferase in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The use of directed mutagenesis, surface plasmon resonance detection and isothermal titration microcalorimetry revealed that several positively charged residues of the PSBD, most notably Arg135, play an important part in the interaction with both E1 and E3, whereas Met131 makes a significant contribution to the binding of E1 only. This indicates that the binding sites for E1 and E3 on the PSBD are overlapping but probably significantly different, and that additional hydrophobic interactions may be involved in binding E1 compared with E3. Arg135 of the PSBD was also replaced with cysteine (R135C), which was then modified chemically by alkylation with increasingly large aliphatic groups (R135C -methyl, -ethyl, -propyl and -butyl). The pattern of changes in the values of ΔG°, ΔH° and TΔS° that were found to accompany the interaction with the variant PSBDs differed between E1 and E3 despite the similarities in the free energies of their binding to the wild-type. The importance of a positive charge on the side-chain at position 135 for the interaction of the PSBD with E3 and E1 was apparent, although lysine was found to be an imperfect substitute for arginine. The results offer further evidence of entropyenthalpy compensation ('thermodynamic homeostasis') - a feature of systems involving a multiplicity of weak interactions.

Original languageEnglish
Pages (from-to)4488-4496
Number of pages9
JournalEuropean Journal of Biochemistry
Volume270
Issue number22
DOIs
Publication statusPublished - 2003 Nov 1

Fingerprint

Dihydrolipoyllysine-Residue Acetyltransferase
Pyruvate Decarboxylase
Multienzyme Complexes
Dihydrolipoamide Dehydrogenase
Pyruvate Dehydrogenase Complex
Geobacillus stearothermophilus
Mutagenesis
Surface Plasmon Resonance
Alkylation
Surface plasmon resonance
Bacilli
Pyruvic Acid
Titration
Hydrophobic and Hydrophilic Interactions
Thermodynamics
Free energy
Lysine
Cysteine
Arginine
Oxidoreductases

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{d0ddb95ac37742efb5dd1b3037f18a2b,
title = "Interactions of the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus",
abstract = "The enzymes pyruvate decarboxylase (E1) and dihydrolipoyl dehydrogenase (E3) bind tightly but in a mutually exclusive manner to the peripheral subunit-binding domain (PSBD) of dihydrolipoyl acetyltransferase in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The use of directed mutagenesis, surface plasmon resonance detection and isothermal titration microcalorimetry revealed that several positively charged residues of the PSBD, most notably Arg135, play an important part in the interaction with both E1 and E3, whereas Met131 makes a significant contribution to the binding of E1 only. This indicates that the binding sites for E1 and E3 on the PSBD are overlapping but probably significantly different, and that additional hydrophobic interactions may be involved in binding E1 compared with E3. Arg135 of the PSBD was also replaced with cysteine (R135C), which was then modified chemically by alkylation with increasingly large aliphatic groups (R135C -methyl, -ethyl, -propyl and -butyl). The pattern of changes in the values of ΔG°, ΔH° and TΔS° that were found to accompany the interaction with the variant PSBDs differed between E1 and E3 despite the similarities in the free energies of their binding to the wild-type. The importance of a positive charge on the side-chain at position 135 for the interaction of the PSBD with E3 and E1 was apparent, although lysine was found to be an imperfect substitute for arginine. The results offer further evidence of entropyenthalpy compensation ('thermodynamic homeostasis') - a feature of systems involving a multiplicity of weak interactions.",
author = "Jung, {Hyo Il} and Alan Cooper and Perham, {Richard N.}",
year = "2003",
month = "11",
day = "1",
doi = "10.1046/j.1432-1033.2003.03842.x",
language = "English",
volume = "270",
pages = "4488--4496",
journal = "FEBS Journal",
issn = "1742-464X",
publisher = "Wiley-Blackwell",
number = "22",

}

TY - JOUR

T1 - Interactions of the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

AU - Jung, Hyo Il

AU - Cooper, Alan

AU - Perham, Richard N.

PY - 2003/11/1

Y1 - 2003/11/1

N2 - The enzymes pyruvate decarboxylase (E1) and dihydrolipoyl dehydrogenase (E3) bind tightly but in a mutually exclusive manner to the peripheral subunit-binding domain (PSBD) of dihydrolipoyl acetyltransferase in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The use of directed mutagenesis, surface plasmon resonance detection and isothermal titration microcalorimetry revealed that several positively charged residues of the PSBD, most notably Arg135, play an important part in the interaction with both E1 and E3, whereas Met131 makes a significant contribution to the binding of E1 only. This indicates that the binding sites for E1 and E3 on the PSBD are overlapping but probably significantly different, and that additional hydrophobic interactions may be involved in binding E1 compared with E3. Arg135 of the PSBD was also replaced with cysteine (R135C), which was then modified chemically by alkylation with increasingly large aliphatic groups (R135C -methyl, -ethyl, -propyl and -butyl). The pattern of changes in the values of ΔG°, ΔH° and TΔS° that were found to accompany the interaction with the variant PSBDs differed between E1 and E3 despite the similarities in the free energies of their binding to the wild-type. The importance of a positive charge on the side-chain at position 135 for the interaction of the PSBD with E3 and E1 was apparent, although lysine was found to be an imperfect substitute for arginine. The results offer further evidence of entropyenthalpy compensation ('thermodynamic homeostasis') - a feature of systems involving a multiplicity of weak interactions.

AB - The enzymes pyruvate decarboxylase (E1) and dihydrolipoyl dehydrogenase (E3) bind tightly but in a mutually exclusive manner to the peripheral subunit-binding domain (PSBD) of dihydrolipoyl acetyltransferase in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The use of directed mutagenesis, surface plasmon resonance detection and isothermal titration microcalorimetry revealed that several positively charged residues of the PSBD, most notably Arg135, play an important part in the interaction with both E1 and E3, whereas Met131 makes a significant contribution to the binding of E1 only. This indicates that the binding sites for E1 and E3 on the PSBD are overlapping but probably significantly different, and that additional hydrophobic interactions may be involved in binding E1 compared with E3. Arg135 of the PSBD was also replaced with cysteine (R135C), which was then modified chemically by alkylation with increasingly large aliphatic groups (R135C -methyl, -ethyl, -propyl and -butyl). The pattern of changes in the values of ΔG°, ΔH° and TΔS° that were found to accompany the interaction with the variant PSBDs differed between E1 and E3 despite the similarities in the free energies of their binding to the wild-type. The importance of a positive charge on the side-chain at position 135 for the interaction of the PSBD with E3 and E1 was apparent, although lysine was found to be an imperfect substitute for arginine. The results offer further evidence of entropyenthalpy compensation ('thermodynamic homeostasis') - a feature of systems involving a multiplicity of weak interactions.

UR - http://www.scopus.com/inward/record.url?scp=0344442892&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0344442892&partnerID=8YFLogxK

U2 - 10.1046/j.1432-1033.2003.03842.x

DO - 10.1046/j.1432-1033.2003.03842.x

M3 - Article

C2 - 14622277

AN - SCOPUS:0344442892

VL - 270

SP - 4488

EP - 4496

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-464X

IS - 22

ER -