PURIFICATION AND CHARACTERIZATION OF AN ALPHA-METHYLACYL-COA RACEMASE FROM HUMAN LIVER

A specific racemase for alpha-methylacyl-CoAs, which had previously been studied in rat liver [W. Schmitz, R. Fingerhut, E. Conzelmann (1994) Eur. J. Biochem. 222, 313-323], has now been demonstrated also in human tissues. The human enzyme cross-reacts with a polyclonal antiserum against the rat liver racemase. The racemase was purified from human liver some 3600-fold. It is a monomer of 47 kDa with an isolectric point of pH 6.1 and is optimally active between pH 7-8. It acts only on coenzyme A thioesters, not on free fatty acids, and accepts as substrates a wide range of alpha-methylacyl-CoAs, including pristanoyl-CoA and trihydroxycoprostanoyl-CoA (an intermediate in bile acid synthesis), but neither 3-methyl-branched nor linear-chain acyl-CoAs. A clear difference in subcellular localization of the enzyme was found between humans and rats: the rat enzyme co-distributed exclusively with mitochondrial marker enzymes whereas in human cells, only 10-30% of the activity was found in mitochondria, the bulk activity was located in peroxisomes. Cells from patients with general deficiency of peroxisome assembly (Zellweger syndrome) showed strongly reduced racemase activity, with only the mitochondrial share being present while the peroxisomal form was absent.