Investigation of the conserved lysines of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase.
Sequence analysis has revealed two classes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Crystal structures of ternary complexes of the Class II enzyme from Pseudomonas mevalonii revealed lysine 267 critically positioned at the active site. This observation suggested a revised catalytic mechanism in which lysine 267 facilitates hydride transfer from reduced coenzyme by polarizing the carbonyl group of HMG-CoA and subsequently of bound mevaldehyde, an inference supported by mutagenesis of lysine 267 to aminoethylcysteine. For this mechanism to be general, Class I HMG-CoA reductases ought also to possess an active site lysine. Three lysines are conserved among all Class I HMG-CoA reductases. The three conserved lysines of Syrian hamster HMG-CoA reductase were mutated to alanine. All three mutant enzymes had reduced but detectable activity. Of the three conserved lysines, sequence alignments implicate lysine 734 of the hamster enzyme as the most likely cognate of P. mevalonii lysine 267. Low activity of enzyme K734A did not reflect an altered structure. Substrate recognition was essentially normal, and both circular dichroism spectroscopy and analytical ultracentrifugation implied a native structure. Enzyme K734A also formed an active heterodimer when coexpressed with inactive mutant enzyme D766N. We infer that a lysine is indeed essential for catalysis by the Class I HMG-CoA reductases and that the revised mechanism for catalysis is general for all HMG-CoA reductases.
Bochar
D A
DA
Departments of Biochemistry and Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
Stauffacher
C V
CV
Rodwell
V W
VW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D006224:Cricetinae; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry* / Q000235:genetics; D008239:Lysine / Q000737:chemistry; D008647:Mesocricetus; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
D006903:Hydroxymethylglutaryl CoA Reductases; D008239:Lysine