HPLC assay for methylmalonyl-CoA epimerase.
Methylmalonyl-CoA epimerase (MCE) is broadly distributed in nature and has diverse cellular roles. Many MCE homologues are represented in public databases, but the biochemical function and physiological roles of the majority of these putative proteins have not been investigated. Here, a simplified assay for MCE is described. In this assay, MCE converted (2S)-methylmalonyl-CoA to (2R)-methylmalonyl-CoA which in turn was converted to succinyl-CoA by methylmalonyl-CoA mutase, an enzyme specific for the 2 R isomer. MCE activity was quantified by measuring the disappearance of methylmalonyl-CoA by HPLC. To obtain the methylmalonyl-CoA mutase which was required as a reagent for the assay, an Escherichia coli strain was constructed that expressed high levels of this enzyme as a fusion protein with an 8x histidine tag. This allowed purification of the mutase in a single affinity chromatography step. Previously reported MCE assays required radioactive substrates and/or multiple reagent enzymes that were difficult to obtain. The assay reported here overcomes these difficulties and hence will facilitate studies of MCEs. Such enzymes play important roles in the metabolism of both prokaryotes and higher eukaryotes including humans.
Bobik
Thomas A
TA
Department of Microbiology and Cell Science, University of Florida, Gainesville 32611, USA. bobik@ufl.edu
Rasche
Madeline E
ME
D000214:Acyl Coenzyme A / Q000378:metabolism; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008722:Methods; D019998:Racemases and Epimerases / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism
D000214:Acyl Coenzyme A; D002413:Cations, Divalent; C015357:methylmalonyl-coenzyme A; D019998:Racemases and Epimerases; C021581:methylmalonyl-coenzyme A racemase