A continuous assay for alpha-methylacyl-coenzyme A racemase using circular dichroism

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Alpha-Methylacyl-coenzyme A racemase (AMACR) is a key enzyme involved in metabolizing branched-chain fatty acids.
• AMACR is recognized as a significant biomarker for prostate cancer.
• The enzyme is a potential therapeutic target for prostate cancer treatment.

Purpose of the Study:

• To develop a continuous circular dichroism (CD)-based assay for measuring AMACR activity.
• To facilitate the development of AMACR inhibitors for prostate cancer therapy.
• To characterize the enzymatic properties of Mycobacterium tuberculosis AMACR (MCR).

Main Methods:

• Subcloning the AMACR gene from Mycobacterium tuberculosis into a pET15b vector.
• Overexpression and purification of the MCR enzyme using metal ion affinity chromatography.
• Monitoring MCR-catalyzed epimerization of ibuprofenoyl-CoA using continuous circular dichroism at 279nm.

Main Results:

• MCR demonstrated higher affinity for (2R)-ibuprofenoyl-CoA but faster turnover for (2S)-ibuprofenoyl-CoA.
• The catalytic efficiency of MCR was reduced when expressed as an N-terminal His(6)-tagged fusion protein.
• The developed CD-based assay provides an economical and efficient alternative to traditional fixed-time assays.

Conclusions:

• The continuous CD-based assay is a viable and efficient method for assessing AMACR activity.
• Understanding MCR kinetics and the impact of N-terminal tags is important for inhibitor development.
• This assay facilitates research into AMACR as a therapeutic target for prostate cancer.