Catalysis by Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Proposed roles of histidine 865, glutamate 558, and aspartate 766.
We employed the overexpressed catalytic domains of wild-type Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) and of mutant enzymes E558Q, H865Q, and D766N to investigate the roles of Glu558, His865, and Asp786 in catalysis. Five reactions were studied: reductive deacylation of HMG-CoA or of mevaldyl-CoA to mevalonate, reduction of mevaldehyde to mevalonate, oxidation of mevaldyl-CoA to HMG-CoA, and oxidative acylation of mevaldehyde to HMG-CoA. While only the wild-type enzyme catalyzed all five reactions, mutant enzymes E558Q and H865Q catalyzed other reactions at significant rates. Mutant enzyme D766N, although apparently structurally similar to the wild-type enzyme, was inactive for all five reactions. While an ineffective catalyst for the overall reaction, mutant enzyme H865Q catalyzed the reduction of mevaldehyde to mevalonate at about the wild-type rate. Coenzyme A, which stimulated mevaldehyde reduction by the wild-type enzyme, inhibited mutant enzyme H865Q, apparently due to an impaired ability to protonate the coenzyme A thioanion, CoAS-. Based on these data, we infer that Glu558, His865, and Asp766 function in catalysis at the indicated stage of the overall reaction, [formula: see text] and we propose a revised mechanism for catalysis by mammalian HMG-CoA reductases.
Frimpong
K
K
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
Rodwell
V W
VW
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D002384:Catalysis; D003001:Cloning, Molecular; D006224:Cricetinae; D005971:Glutamates; D018698:Glutamic Acid; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008647:Mesocricetus; D008962:Models, Theoretical; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000302:isolation & purification; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D005971:Glutamates; D010446:Peptide Fragments; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D006903:Hydroxymethylglutaryl CoA Reductases