A high-performance liquid chromatography assay for threonine/serine dehydratase

Area of Science:

• Biochemistry
• Enzymology
• Analytical Chemistry

Background:

• Threonine dehydratase is crucial for isoleucine biosynthesis.
• Existing assays for threonine dehydratase lack sensitivity and product identification capabilities.

Purpose of the Study:

• To develop a highly sensitive and specific assay for threonine dehydratase activity.
• To enable the identification of reaction products using HPLC.

Main Methods:

• Development of a novel uv/fluorometric High-Performance Liquid Chromatography (HPLC) assay.
• Enzyme reaction product derivatization using o-phenylenediamine.
• Spectrophotometric or fluorometric detection of derivatized 2-ketoacids.

Main Results:

• The novel HPLC assay demonstrated over 1000-fold greater sensitivity compared to the dinitrophenyl hydrazine assay.
• The assay successfully identified reaction products, distinguishing between different ketoacids.
• The method is applicable to analyzing enzyme kinetics with mixed substrates like threonine and serine.

Conclusions:

• A sensitive and specific HPLC assay for threonine dehydratase has been established.
• This assay significantly advances the analytical capabilities for studying isoleucine biosynthesis pathways.
• The method provides a robust tool for enzyme characterization and kinetic studies.