Purification and properties of an alpha-methylacyl-CoA racemase from rat liver.
The (R)- and (S)-isomers of alpha-methyl-branched fatty acids were shown to be rapidly interconverted as coenzyme A thioesters, by an alpha-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 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. The racemase catalyzes a rapid exchange of the H atom in the alpha-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-(3)H]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.
Schmitz
W
W
Theodor-Boveri-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Germany.
Fingerhut
R
R
Conzelmann
E
E
D000214:Acyl Coenzyme A / Q000378:metabolism; D000818:Animals; D002264:Carboxylic Acids / Q000138:chemical synthesis; D002845:Chromatography; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000494:pharmacology; D017886:Durapatite; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D019998:Racemases and Epimerases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D013237:Stereoisomerism; D013379:Substrate Specificity; D014316:Tritium; D014462:Ultrafiltration
D000214:Acyl Coenzyme A; D002264:Carboxylic Acids; D003902:Detergents; D007202:Indicators and Reagents; D014316:Tritium; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D017886:Durapatite; D019998:Racemases and Epimerases; C108352:alpha-methylacyl-CoA racemase