The active site of hamster 3-hydroxy-3-methylglutaryl-CoA reductase resides at the subunit interface and incorporates catalytically essential acidic residues from separate polypeptides.
We employed site-directed mutagenesis based on sequence comparisons and characterization of purified mutant enzymes to identify Glu558 and Asp766 of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) as essential for catalysis. Mutant enzymes E558D, E558Q, and D766N had wild-type Km values for (S)-HMG-CoA and NADPH, but exhibited less than 0.5% of the wild-type catalytic activity. The inactive mutant polypeptides E558Q and D766N nevertheless can associate to generate an active enzyme. In vitro, 6% of the wild-type activity was observed when mutant polypeptides E558D and D766N were mixed in the absence of chaotropic agents. When mutant polypeptides E558Q and D766N were co-expressed in Escherichia coli, the resulting purified enzyme had 25% of wild-type activity. Hamster HMG-CoA reductase thus is a two-site, dimeric enzyme whose subunits associate to form an active site in which each monomer contributes at least one residue (e.g. Glu558 from one monomer and Asp766 from the other). The wild-type enzyme behaves as a dimer during size exclusion chromatography and has one HMG-CoA binding site per monomer. Syrian hamster HMG-CoA reductase thus appears to be a homodimer with two active sites which are located at the subunit interface.
Frimpong
K
K
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
Rodwell
V W
VW
D000214:Acyl Coenzyme A / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D006224:Cricetinae; D005816:Genetic Complementation Test; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry*; D007700:Kinetics; D046911:Macromolecular Substances; D008647:Mesocricetus; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011487:Protein Conformation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D000214:Acyl Coenzyme A; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; C008047:3-hydroxy-3-methylglutaryl-coenzyme A; D006903:Hydroxymethylglutaryl CoA Reductases