title: Purification and properties of the catalytic domain of human 3-hydroxy-3-methylglutaryl-CoA reductase expressed in Escherichia coli.
abstract: Three fragments of the cDNA encoding human 3-hydroxy-3-methylglutaryl-CoA reductase, all incorporating the majority of the catalytic domain of the protein, were subcloned into Escherichia coli expression vectors containing the pL promoter. The two larger expressed fragments (58 and 52 kDa) were soluble and had enzymatic activity, while the smallest (48 kDa) was insoluble. The two active fragments were purified by a combination of conventional techniques and affinity chromatography. A number of properties of the two enzymes were compared including specific activity, kinetic parameters, relative solubility, and cold lability. The 52-kDa enzyme was observed to change from a dimeric to monomeric form and to lose activity at 4 degrees C. In contrast, the 58-kDa enzyme was found to be much less cold labile, and was dimeric at both 20 and 4 degrees C. In order to resolve the number of subunits required to form an active site, the number of inhibitor binding sites for a known inhibitor was determined to be one per subunit in the 58-kDa enzyme.
authors.affiliation: Department of Medicinal Chemistry, Smith Kline & French Laboratories, Swedeland, Pennsylvania 19479.
authors.forename: R J
authors.initials: RJ
authors.lastname: Mayer
authors.forename: C
authors.initials: C
authors.lastname: Debouck
authors.forename: B W
authors.initials: BW
authors.lastname: Metcalf
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012995:Solubility
chemical_list: D010446:Peptide Fragments; D004247:DNA; D006903:Hydroxymethylglutaryl CoA Reductases