Purification and properties of an α‐methylacyl‐CoA racemase from rat liver

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Abstract
The (R)- and (S)-isomers of α-methyl-branched fatty acids were shown to be rapidly interconverted as coenzyme A thioesters, by an α-methylacyl-CoA racemase. The enzyme was purified some 5600-fold from rat liver, to apparent homogeneity. It is a monomer of 45 kDa with an isolectric point of pH 6.1 and is optimally active between pH 6 and pH 7. It acts only on coenzyme A thioesters, not on free fatty acids, and accepts as substrates a wide range of α-methylacyl-CoAs, including pristanoyl-CoA and trihydroxycoprostanoyl-CoA (an intermediate in bile acid synthesis), but neither 3-methyl-branched nor linear-chain acyl-CoAs. The racemase catalyzes a rapid exchange of the H atom in the α-position of the fatty acid against a proton from water, indicating that the mechanism involves abstraction of a proton. Based on this observation, a very sensitive and convenient radiometric assay, with 2-methyl[2-3H]acyl-CoAs as substrates, was developed. The enzyme was inactivated by micromolar concentrations of Hg2+ and to a lesser extent by Cu2+ but not by iodoacetamide and only slightly by N-ethylmaleimide and thimerosal.