Sequential domain refolding of pig muscle 3-phosphoglycerate kinase: Kinetic analysis of reactivation

Andrea N. Szilágyi, M. Vas

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Background: Slow refolding of 3-phosphoglycerate kinase is supposed to be caused mainly by its domain structure: folding of the C-terminal domain and/or domain pairing has been suggested to be the rate-limiting step. A slow isomerization has been observed during refolding of the isolated C-terminal proteolytic fragment (larger than the C-domain of about 22 kDa by 5 kDa) of the pig muscle enzyme. Here, the role of this step in the reformation of the active enzyme species is investigated. Results: The time course of reactivation during refolding of 3-phosphoglycerate kinase or its complementary proteolytic fragments (residues 1-155 and 156-416) exhibits a pronounced lag-phase indicating the formation of an inactive folding intermediate. The whole process, which leads to a high (60-85%) recovery of the enzyme activity, can be described by two consecutive first-order steps (with rate constants 0.012 ± 0.0035 and 0.007 ± 0.0020 s-1). A prior renaturation of the c-fragment restores MgATP binding by the C-domain and abolishes the faster step, allowing the separate observation of the slower step. In accordance with this, refolding of the C-domain as monitored by a change in Trp fluorescence occurs at a rate similar to that of the faster step. Conclusions: In addition to the previously observed slow refolding step (0.012 s-1) within the C-domain, the occurrence of another slow step (0.007 s-1), probably within the N-domain, is detected. The independence of the folding of the C-domain is demonstrated whereas, from the comparative kinetic analysis, independent folding of the N-domain looks less probable. Our data are more compatible with a sequential, rather than random, mechanism and suggest that folding of the C-domain, leading to an inactive intermediate, occurs first, followed by folding of the N-domain.

Original languageEnglish
Pages (from-to)565-575
Number of pages11
JournalFolding and Design
Volume3
Issue number6
DOIs
Publication statusPublished - 1998

Fingerprint

Phosphoglycerate Kinase
Muscle
Swine
Muscles
Kinetics
Enzyme activity
Enzymes
Isomerization
Rate constants
Adenosine Triphosphate
Fluorescence
Recovery
Observation
3-phosphoglycerate

Keywords

  • 3-phosphoglycerate kinase
  • Complementary fragments
  • Domains
  • Protein folding
  • Reactivation kinetics

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine

Cite this

Sequential domain refolding of pig muscle 3-phosphoglycerate kinase : Kinetic analysis of reactivation. / Szilágyi, Andrea N.; Vas, M.

In: Folding and Design, Vol. 3, No. 6, 1998, p. 565-575.

Research output: Contribution to journalArticle

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abstract = "Background: Slow refolding of 3-phosphoglycerate kinase is supposed to be caused mainly by its domain structure: folding of the C-terminal domain and/or domain pairing has been suggested to be the rate-limiting step. A slow isomerization has been observed during refolding of the isolated C-terminal proteolytic fragment (larger than the C-domain of about 22 kDa by 5 kDa) of the pig muscle enzyme. Here, the role of this step in the reformation of the active enzyme species is investigated. Results: The time course of reactivation during refolding of 3-phosphoglycerate kinase or its complementary proteolytic fragments (residues 1-155 and 156-416) exhibits a pronounced lag-phase indicating the formation of an inactive folding intermediate. The whole process, which leads to a high (60-85{\%}) recovery of the enzyme activity, can be described by two consecutive first-order steps (with rate constants 0.012 ± 0.0035 and 0.007 ± 0.0020 s-1). A prior renaturation of the c-fragment restores MgATP binding by the C-domain and abolishes the faster step, allowing the separate observation of the slower step. In accordance with this, refolding of the C-domain as monitored by a change in Trp fluorescence occurs at a rate similar to that of the faster step. Conclusions: In addition to the previously observed slow refolding step (0.012 s-1) within the C-domain, the occurrence of another slow step (0.007 s-1), probably within the N-domain, is detected. The independence of the folding of the C-domain is demonstrated whereas, from the comparative kinetic analysis, independent folding of the N-domain looks less probable. Our data are more compatible with a sequential, rather than random, mechanism and suggest that folding of the C-domain, leading to an inactive intermediate, occurs first, followed by folding of the N-domain.",
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AU - Szilágyi, Andrea N.

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N2 - Background: Slow refolding of 3-phosphoglycerate kinase is supposed to be caused mainly by its domain structure: folding of the C-terminal domain and/or domain pairing has been suggested to be the rate-limiting step. A slow isomerization has been observed during refolding of the isolated C-terminal proteolytic fragment (larger than the C-domain of about 22 kDa by 5 kDa) of the pig muscle enzyme. Here, the role of this step in the reformation of the active enzyme species is investigated. Results: The time course of reactivation during refolding of 3-phosphoglycerate kinase or its complementary proteolytic fragments (residues 1-155 and 156-416) exhibits a pronounced lag-phase indicating the formation of an inactive folding intermediate. The whole process, which leads to a high (60-85%) recovery of the enzyme activity, can be described by two consecutive first-order steps (with rate constants 0.012 ± 0.0035 and 0.007 ± 0.0020 s-1). A prior renaturation of the c-fragment restores MgATP binding by the C-domain and abolishes the faster step, allowing the separate observation of the slower step. In accordance with this, refolding of the C-domain as monitored by a change in Trp fluorescence occurs at a rate similar to that of the faster step. Conclusions: In addition to the previously observed slow refolding step (0.012 s-1) within the C-domain, the occurrence of another slow step (0.007 s-1), probably within the N-domain, is detected. The independence of the folding of the C-domain is demonstrated whereas, from the comparative kinetic analysis, independent folding of the N-domain looks less probable. Our data are more compatible with a sequential, rather than random, mechanism and suggest that folding of the C-domain, leading to an inactive intermediate, occurs first, followed by folding of the N-domain.

AB - Background: Slow refolding of 3-phosphoglycerate kinase is supposed to be caused mainly by its domain structure: folding of the C-terminal domain and/or domain pairing has been suggested to be the rate-limiting step. A slow isomerization has been observed during refolding of the isolated C-terminal proteolytic fragment (larger than the C-domain of about 22 kDa by 5 kDa) of the pig muscle enzyme. Here, the role of this step in the reformation of the active enzyme species is investigated. Results: The time course of reactivation during refolding of 3-phosphoglycerate kinase or its complementary proteolytic fragments (residues 1-155 and 156-416) exhibits a pronounced lag-phase indicating the formation of an inactive folding intermediate. The whole process, which leads to a high (60-85%) recovery of the enzyme activity, can be described by two consecutive first-order steps (with rate constants 0.012 ± 0.0035 and 0.007 ± 0.0020 s-1). A prior renaturation of the c-fragment restores MgATP binding by the C-domain and abolishes the faster step, allowing the separate observation of the slower step. In accordance with this, refolding of the C-domain as monitored by a change in Trp fluorescence occurs at a rate similar to that of the faster step. Conclusions: In addition to the previously observed slow refolding step (0.012 s-1) within the C-domain, the occurrence of another slow step (0.007 s-1), probably within the N-domain, is detected. The independence of the folding of the C-domain is demonstrated whereas, from the comparative kinetic analysis, independent folding of the N-domain looks less probable. Our data are more compatible with a sequential, rather than random, mechanism and suggest that folding of the C-domain, leading to an inactive intermediate, occurs first, followed by folding of the N-domain.

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KW - Complementary fragments

KW - Domains

KW - Protein folding

KW - Reactivation kinetics

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