Monitoring the acetohydroxy acid synthase reaction and related carboligations by circular dichroism spectroscopy

Michael Vinogradov, Alexander Kaplun, Maria Vyazmensky, Stanislav Engel, Ralph Golbik, Kai Tittmann, Kathrin Uhlemann, Ludmilla Meshalkina, Ze'ev Barak, Gerhard Hübner, David M. Chipman

Research output: Contribution to journalArticlepeer-review

20 Scopus citations

Abstract

Acetohydroxy acid synthase (AHAS) and related enzymes catalyze the production of chiral compounds [(S)-acetolactate, (S)-acetohydroxybutyrate, or (R)-phenylacetylcarbinol] from achiral substrates (pyruvate, 2-ketobutyrate, or benzaldehyde). The common methods for the determination of AHAS activity have shortcomings. The colorimetric method for detection of acyloins formed from the products is tedious and does not allow time-resolved measurements. The continuous assay for consumption of pyruvate based on its absorbance at 333 nm, though convenient, is limited by the extremely small extinction coefficient of pyruvate, which results in a low signal-to-noise ratio and sensitivity to interfering absorbing compounds. Here, we report the use of circular dichroism spectroscopy for monitoring AHAS activity. This method, which exploits the optical activity of reaction products, displays a high signal-to-noise ratio and is easy to perform both in time-resolved and in commercial modes. In addition to AHAS, we examined the determination of activity of glyoxylate carboligase. This enzyme catalyzes the condensation of two molecules of glyoxylate to chiral tartronic acid semialdehyde. The use of circular dichroism also identifies the product of glyoxylate carboligase as being in the (R) configuration.

Original languageEnglish
Pages (from-to)126-133
Number of pages8
JournalAnalytical Biochemistry
Volume342
Issue number1
DOIs
StatePublished - 1 Jul 2005

Keywords

  • Acetohydroxy acid synthase
  • Acetohydroxybutyrate
  • Acetolactate
  • Circular dichroism
  • Glyoxylate carboligase
  • Phenylacetylcarbinol
  • Tartronate semialdehyde

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint

Dive into the research topics of 'Monitoring the acetohydroxy acid synthase reaction and related carboligations by circular dichroism spectroscopy'. Together they form a unique fingerprint.

Cite this