Correlation between conformational stability of the ternary enzyme-substrate complex and domain closure of 3-phosphoglycerate kinase

A. Varga, Flachner Beáta, Éva Gráczer, Szabolcs Osváth, Andrea N. Szilágyi, M. Vas

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

3-Phosphoglycerate kinase (PGK) is a typical two-domain hinge-bending enzyme with a well-structured interdomain region. The mechanism of domain-domain interaction and its regulation by substrate binding is not yet fully understood. Here the existence of strong cooperativity between the two domains was demonstrated by following heat transitions of pig muscle and yeast PGKs using differential scanning microcalorimetry and fluorimetry. Two mutants of yeast PGK containing a single tryptophan fluorophore either in the N- or in the C-terminal domain were also studied. The coincidence of the calorimetric and fluorimetric heat transitions in all cases indicated simultaneous, highly cooperative unfolding of the two domains. This cooperativity is preserved in the presence of substrates: 3-phosphoglycerate bound to the N domain or the nucleotide (MgADP, MgATP) bound to the C domain increased the structural stability of the whole molecule. A structural explanation of domain-domain interaction is suggested by analysis of the atomic contacts in 12 different PGK crystal structures. Well-defined backbone and side-chain H bonds, and hydrophobic and electrostatic interactions between side chains of conserved residues are proposed to be responsible for domain-domain communication. Upon binding of each substrate newly formed molecular contacts are identified that firstly explain the order of the increased heat stability in the various binary complexes, and secondly describe the possible route of transmission of the substrate-induced conformational effects from one domain to the other. The largest stability is characteristic of the native ternary complex and is abolished in the case of a chemically modified inactive form of PGK, the domain closure of which was previously shown to be prevented [Sinev MA, Razgulyaev OI, Vas M, Timchenko AA & Ptitsyn OB (1989) Eur J Biochem 180, 61-66]. Thus, conformational stability correlates with domain closure that requires simultaneous binding of both substrates.

Original languageEnglish
Pages (from-to)1867-1885
Number of pages19
JournalFEBS Journal
Volume272
Issue number8
DOIs
Publication statusPublished - Apr 2005

Fingerprint

Phosphoglycerate Kinase
Enzyme Stability
Substrates
Enzymes
Hot Temperature
Yeast
Yeasts
Fluorometry
Fluorophores
Hinges
Coulomb interactions
Static Electricity
Hydrophobic and Hydrophilic Interactions
Tryptophan
Adenosine Diphosphate
Muscle
Swine
Nucleotides
Adenosine Triphosphate
Crystal structure

Keywords

  • Domain closure
  • Molecular graphics
  • Phosphoglycerate kinase
  • Substrate effect
  • Thermal unfolding

ASJC Scopus subject areas

  • Biochemistry

Cite this

Correlation between conformational stability of the ternary enzyme-substrate complex and domain closure of 3-phosphoglycerate kinase. / Varga, A.; Beáta, Flachner; Gráczer, Éva; Osváth, Szabolcs; Szilágyi, Andrea N.; Vas, M.

In: FEBS Journal, Vol. 272, No. 8, 04.2005, p. 1867-1885.

Research output: Contribution to journalArticle

Varga, A. ; Beáta, Flachner ; Gráczer, Éva ; Osváth, Szabolcs ; Szilágyi, Andrea N. ; Vas, M. / Correlation between conformational stability of the ternary enzyme-substrate complex and domain closure of 3-phosphoglycerate kinase. In: FEBS Journal. 2005 ; Vol. 272, No. 8. pp. 1867-1885.
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KW - Substrate effect

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