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 co enzyme A (HMG-CoA) reductase. Crystal structures of ternary complexes of th e Class II enzyme from Pseudomonas mevalonii revealed lysine 267 critically positioned at the active site. This observation suggested a revised cataly tic mechanism in which lysine 267 facilitates hydride transfer from reduced coenzyme by polarizing the carbonyl group of HMG-CoA and subsequently of b ound mevaldehyde, an inference supported by mutagenesis of lysine 267 to am inoethylcysteine. For this mechanism to be general, Class I HMG-CoA reducta ses ought also to possess an active site lysine. Three lysines are conserve d among all Class I HMG-CoA reductases. The three conserved lysines of Syri an hamster HMG-CoA reductase were mutated to alanine. All three mutant enzy mes had reduced but detectable activity. Of the three conserved lysines, se quence alignments implicate lysine 734 of the hamster enzyme as the most li kely cognate of P. mevalonii lysine 267. Low activity of enzyme K734A did n ot reflect an altered structure. Substrate recognition was essentially norm al, and both circular dichroism spectroscopy and analytical ultracentrifuga tion implied a native structure. Enzyme K734A also formed an active heterod imer when coexpressed with inactive mutant enzyme D766N. We infer that a ly sine is indeed essential for catalysis by the Class I HMG-CoA reductases an d that the revised mechanism for catalysis is general for all HMG-CoA reduc tases.