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Subunit association in acetohydroxy acid synthase isozyme III

C Sella1, O Weinstock, Z Barak

  • 1Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.

Journal of Bacteriology
|September 1, 1993
PubMed
Summary

Acetohydroxy acid synthase isozyme III (AHAS III) from E. coli dissociates easily, requiring excess small subunits for accurate activity measurement. Valine likely inhibits the holoenzyme, not by causing dissociation.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • Acetohydroxy acid synthase isozyme III (AHAS III) from Escherichia coli is a heterotetrameric enzyme (α2β2) crucial for amino acid biosynthesis.
  • The enzyme consists of a large catalytic subunit (α) and a small regulatory subunit (β), encoded by ilvI and ilvH genes, respectively.

Purpose of the Study:

  • To investigate the subunit interactions and dissociation characteristics of E. coli AHAS III.
  • To understand the implications of subunit dissociation on enzyme activity measurements and valine sensitivity.

Main Methods:

  • Purification of AHAS III enzyme from E. coli.
  • Analysis of subunit interactions using purified enzyme and subcloned subunits.
  • Enzyme activity assays under varying subunit concentrations and valine inhibition studies.

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Main Results:

  • AHAS III exhibits reversible subunit dissociation with a high dissociation constant, indicating instability in its holoenzyme form.
  • Enzyme activity is concentration-dependent and underestimated in extracts unless excess small subunits are added to stabilize the holoenzyme.
  • Valine inhibition is likely mediated by binding to the intact holoenzyme (I2H2), independent of subunit dissociation.

Conclusions:

  • The high dissociation constant of AHAS III subunits necessitates specific conditions for accurate activity assessment in vitro and in vivo.
  • AHAS III is the predominant isozyme in E. coli under certain growth conditions, and its activity is sensitive to experimental procedures.
  • Unlike other isozymes, AHAS III shows a significant tendency to dissociate, impacting its functional and regulatory properties.