The N-terminal domain of the regulatory subunit is sufficient for complete activation of acetohydroxyacid synthase III from Escherichia coli

Sharon Mendel, Michael Vinogradov, Maria Vyazmensky, David M Chipman, Ze'ev Barak

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

We have previously proposed a model for the fold of the N-terminal domain of the small, regulatory subunit (SSU) of acetohydroxyacid synthase isozyme III. The fold is an alpha-beta sandwich with betaalphabetabetaalphabeta topology, structurally homologous to the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase. We suggested that the N-terminal domains of a pair of SSUs interact in the holoenzyme to form two binding sites for the feedback inhibitor valine in the interface between them. The model was supported by mutational analysis and other evidence. We have now examined the role of the C-terminal portion of the SSU by construction of truncated polypeptides (lacking 35, 48, 80, 95, or 112 amino acid residues from the C terminus) and examining the properties of holoenzymes reconstituted using these constructs. The Delta35, Delta48, and Delta80 constructs all lead to essentially complete activation of the catalytic subunits. The Delta80 construct, corresponding to the putative N-terminal domain, has the highest level of affinity for the catalytic subunits and leads to a reconstituted enzyme with k(cat)/K(M) about twice that of the wild-type enzyme. On the other hand, none of these constructs binds valine or leads to a valine-sensitive enzyme on reconstitution. The enzyme reconstituted with the Delta80 construct does not bind valine, either. The N-terminal portion (about 80 amino acid residues) of the SSU is thus necessary and sufficient for recognition and activation of the catalytic subunits, but the C-terminal half of the SSU is required for valine binding and response. We suggest that the C-terminal region of the SSU contributes to monomer-monomer interactions, and provide additional experimental evidence for this suggestion.

Original languageEnglish
Pages (from-to)275-284
Number of pages10
JournalJournal of Molecular Biology
Volume325
Issue number2
Early online date11 Dec 2002
DOIs
Publication statusPublished - 10 Jan 2003

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Valine
Escherichia coli
Catalytic Domain
Holoenzymes
Enzymes
Phosphoglycerate Dehydrogenase
Amino Acids
Nucleic Acid Regulatory Sequences
Isoenzymes
Binding Sites
Peptides

Keywords

  • Acetolactate Synthase
  • Amino Acid Sequence
  • Enzyme Activation
  • Escherichia coli
  • Escherichia coli Proteins
  • Fluorescence Resonance Energy Transfer
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Folding
  • Protein Structure, Tertiary
  • Protein Subunits
  • Valine
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

The N-terminal domain of the regulatory subunit is sufficient for complete activation of acetohydroxyacid synthase III from Escherichia coli. / Mendel, Sharon; Vinogradov, Michael; Vyazmensky, Maria; Chipman, David M; Barak, Ze'ev.

In: Journal of Molecular Biology, Vol. 325, No. 2, 10.01.2003, p. 275-284.

Research output: Contribution to journalArticle

Mendel, Sharon ; Vinogradov, Michael ; Vyazmensky, Maria ; Chipman, David M ; Barak, Ze'ev. / The N-terminal domain of the regulatory subunit is sufficient for complete activation of acetohydroxyacid synthase III from Escherichia coli. In: Journal of Molecular Biology. 2003 ; Vol. 325, No. 2. pp. 275-284.
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abstract = "We have previously proposed a model for the fold of the N-terminal domain of the small, regulatory subunit (SSU) of acetohydroxyacid synthase isozyme III. The fold is an alpha-beta sandwich with betaalphabetabetaalphabeta topology, structurally homologous to the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase. We suggested that the N-terminal domains of a pair of SSUs interact in the holoenzyme to form two binding sites for the feedback inhibitor valine in the interface between them. The model was supported by mutational analysis and other evidence. We have now examined the role of the C-terminal portion of the SSU by construction of truncated polypeptides (lacking 35, 48, 80, 95, or 112 amino acid residues from the C terminus) and examining the properties of holoenzymes reconstituted using these constructs. The Delta35, Delta48, and Delta80 constructs all lead to essentially complete activation of the catalytic subunits. The Delta80 construct, corresponding to the putative N-terminal domain, has the highest level of affinity for the catalytic subunits and leads to a reconstituted enzyme with k(cat)/K(M) about twice that of the wild-type enzyme. On the other hand, none of these constructs binds valine or leads to a valine-sensitive enzyme on reconstitution. The enzyme reconstituted with the Delta80 construct does not bind valine, either. The N-terminal portion (about 80 amino acid residues) of the SSU is thus necessary and sufficient for recognition and activation of the catalytic subunits, but the C-terminal half of the SSU is required for valine binding and response. We suggest that the C-terminal region of the SSU contributes to monomer-monomer interactions, and provide additional experimental evidence for this suggestion.",
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T1 - The N-terminal domain of the regulatory subunit is sufficient for complete activation of acetohydroxyacid synthase III from Escherichia coli

AU - Mendel, Sharon

AU - Vinogradov, Michael

AU - Vyazmensky, Maria

AU - Chipman, David M

AU - Barak, Ze'ev

PY - 2003/1/10

Y1 - 2003/1/10

N2 - We have previously proposed a model for the fold of the N-terminal domain of the small, regulatory subunit (SSU) of acetohydroxyacid synthase isozyme III. The fold is an alpha-beta sandwich with betaalphabetabetaalphabeta topology, structurally homologous to the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase. We suggested that the N-terminal domains of a pair of SSUs interact in the holoenzyme to form two binding sites for the feedback inhibitor valine in the interface between them. The model was supported by mutational analysis and other evidence. We have now examined the role of the C-terminal portion of the SSU by construction of truncated polypeptides (lacking 35, 48, 80, 95, or 112 amino acid residues from the C terminus) and examining the properties of holoenzymes reconstituted using these constructs. The Delta35, Delta48, and Delta80 constructs all lead to essentially complete activation of the catalytic subunits. The Delta80 construct, corresponding to the putative N-terminal domain, has the highest level of affinity for the catalytic subunits and leads to a reconstituted enzyme with k(cat)/K(M) about twice that of the wild-type enzyme. On the other hand, none of these constructs binds valine or leads to a valine-sensitive enzyme on reconstitution. The enzyme reconstituted with the Delta80 construct does not bind valine, either. The N-terminal portion (about 80 amino acid residues) of the SSU is thus necessary and sufficient for recognition and activation of the catalytic subunits, but the C-terminal half of the SSU is required for valine binding and response. We suggest that the C-terminal region of the SSU contributes to monomer-monomer interactions, and provide additional experimental evidence for this suggestion.

AB - We have previously proposed a model for the fold of the N-terminal domain of the small, regulatory subunit (SSU) of acetohydroxyacid synthase isozyme III. The fold is an alpha-beta sandwich with betaalphabetabetaalphabeta topology, structurally homologous to the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase. We suggested that the N-terminal domains of a pair of SSUs interact in the holoenzyme to form two binding sites for the feedback inhibitor valine in the interface between them. The model was supported by mutational analysis and other evidence. We have now examined the role of the C-terminal portion of the SSU by construction of truncated polypeptides (lacking 35, 48, 80, 95, or 112 amino acid residues from the C terminus) and examining the properties of holoenzymes reconstituted using these constructs. The Delta35, Delta48, and Delta80 constructs all lead to essentially complete activation of the catalytic subunits. The Delta80 construct, corresponding to the putative N-terminal domain, has the highest level of affinity for the catalytic subunits and leads to a reconstituted enzyme with k(cat)/K(M) about twice that of the wild-type enzyme. On the other hand, none of these constructs binds valine or leads to a valine-sensitive enzyme on reconstitution. The enzyme reconstituted with the Delta80 construct does not bind valine, either. The N-terminal portion (about 80 amino acid residues) of the SSU is thus necessary and sufficient for recognition and activation of the catalytic subunits, but the C-terminal half of the SSU is required for valine binding and response. We suggest that the C-terminal region of the SSU contributes to monomer-monomer interactions, and provide additional experimental evidence for this suggestion.

KW - Acetolactate Synthase

KW - Amino Acid Sequence

KW - Enzyme Activation

KW - Escherichia coli

KW - Escherichia coli Proteins

KW - Fluorescence Resonance Energy Transfer

KW - Molecular Sequence Data

KW - Mutagenesis, Site-Directed

KW - Protein Folding

KW - Protein Structure, Tertiary

KW - Protein Subunits

KW - Valine

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1016/S0022-2836(02)01142-7

DO - 10.1016/S0022-2836(02)01142-7

M3 - Article

VL - 325

SP - 275

EP - 284

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 2

ER -