A continuous assay for alpha-methylacyl-coenzyme A racemase using circular dichroism.
alpha-Methylacyl-coenzyme A racemase (AMACR) catalyzes the epimerization of (2R)- and (2S)-methyl branched fatty acyl-coenzyme A (CoA) thioesters. AMACR is a biomarker for prostate cancer and a putative target for the development of therapeutic agents directed against the disease. To facilitate development of AMACR inhibitors, a continuous circular dichroism (CD)-based assay has been developed. The open reading frame encoding AMACR from Mycobacterium tuberculosis (MCR) was subcloned into a pET15b vector, and the enzyme was overexpressed and purified using metal ion affinity chromatography. The rates of MCR-catalyzed epimerization of either (2R)- or (2S)-ibuprofenoyl-CoA were determined by following the change in ellipticity at 279nm in the presence of octyl-beta-d-glucopyranoside (0.2%). MCR exhibited slightly higher affinity for (2R)-ibuprofenoyl-CoA (K(m)=48+/-5microM, k(cat)=291+/-30s(-1)), but turned over (2S)-ibuprofenoyl-CoA (K(m)=86+/-6microM, k(cat)=450+/-14s(-1)) slightly faster. MCR expressed as a fusion protein bearing an N-terminal His(6)-tag had a catalytic efficiency (k(cat)/K(m)) that was reduced 22% and 47% in the 2S-->2R and 2R-->2S directions, respectively, relative to untagged enzyme. The continuous CD-based assay offers an economical and efficient alternative method to the labor-intensive, fixed-time assays currently used to measure AMACR activity.
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5.
Dahmane
D
Ouazia
Stephen L
SL
Bearne
D000214:Acyl Coenzyme A / Q000378:metabolism; D002942:Circular Dichroism / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008297:Male; D011471:Prostatic Neoplasms / Q000378:metabolism; D019998:Racemases and Epimerases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism
D000214:Acyl Coenzyme A; D011994:Recombinant Proteins; C070652:ibuprofenyl-coenzyme A; D019998:Racemases and Epimerases; C108352:alpha-methylacyl-CoA racemase