TY - JOUR
T1 - Monitoring the acetohydroxy acid synthase reaction and related carboligations by circular dichroism spectroscopy
AU - Vinogradov, Michael
AU - Kaplun, Alexander
AU - Vyazmensky, Maria
AU - Engel, Stanislav
AU - Golbik, Ralph
AU - Tittmann, Kai
AU - Uhlemann, Kathrin
AU - Meshalkina, Ludmilla
AU - Barak, Ze'ev
AU - Hübner, Gerhard
AU - Chipman, David M.
N1 - Funding Information:
We thank Valery Vinogradov for a generous gift of the purified AHAS I. The work reported here was supported in part by research grants from the Israel Science Foundation to D.M.C. (Grant 660/01), and to Z.B. (467/02), by a seed grant to D.M.C. from the Vice-President for Research and Development at Ben-Gurion University, and by the Deutsche Forschungsgemeinschaft (Germany) and the Fonds der Chemischen Industrie (Germany).
PY - 2005/7/1
Y1 - 2005/7/1
N2 - 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.
AB - 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.
KW - Acetohydroxy acid synthase
KW - Acetohydroxybutyrate
KW - Acetolactate
KW - Circular dichroism
KW - Glyoxylate carboligase
KW - Phenylacetylcarbinol
KW - Tartronate semialdehyde
UR - http://www.scopus.com/inward/record.url?scp=20444426493&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2005.03.049
DO - 10.1016/j.ab.2005.03.049
M3 - Article
AN - SCOPUS:20444426493
SN - 0003-2697
VL - 342
SP - 126
EP - 133
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -