Monitoring the acetohydroxy acid synthase reaction and related carboligations by circular dichroism spectroscopy.
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.
Vinogradov
Michael
M
Department of Life Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva, 84105, Israel.
Kaplun
Alexander
A
Vyazmensky
Maria
M
Engel
Stanislav
S
Golbik
Ralph
R
Tittmann
Kai
K
Uhlemann
Kathrin
K
Meshalkina
Ludmilla
L
Barak
Ze'ev
Z
Hübner
Gerhard
G
Chipman
David M
DM
D000095:Acetolactate Synthase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D002262:Carboxy-Lyases / Q000378:metabolism; D002942:Circular Dichroism / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology; D006038:Glyoxylates / Q000494:pharmacology; D007773:Lactates / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism; D013237:Stereoisomerism; D014633:Valine / Q000494:pharmacology
D006038:Glyoxylates; D007773:Lactates; C006359:alpha-acetolactate; D019289:Pyruvic Acid; D000095:Acetolactate Synthase; D002262:Carboxy-Lyases; C022713:tartronate-semialdehyde synthase; C023746:acetolactate decarboxylase; D014633:Valine; C031150:glyoxylic acid