3-Hydroxy-3-methylglutaryl-coenzyme A reductase of Haloferax volcanii: role of histidine 398 and attenuation of activity by introduction of negative charge at position 404.
Mutant 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases of the halophilic archaeon Haloferax volcanii were constructed to test the proposed mechanism that phosphorylation downregulates the activity of higher eukarya HMG-CoA reductases via charge-charge interaction with the active site histidine. To first verify the sequence-based inference that His 398 is the catalytic histidine of the H. volcanii enzyme, enzyme H398Q was constructed, purified, and assayed for catalysis of three reactions: [1] reductive deacylation of HMG-CoA, [2] reduction of mevaldehyde, and [3] oxidative acylation of mevaldehyde. Enzyme H398Q had low activity for catalysis of reaction [1] or [3], but readily catalyzed mevaldehyde reduction. By analogy to hamster HMG-CoA reductase, we conclude that His 398 is the active site histidine. Mutant forms of the 403-residue H. volcanii enzyme were constructed to model phosphorylation and infer whether attenuated activity involved interaction with His 398. Chimeric H. volcanii-hamster enzymes constructed in an effort to create an active, phosphorylatable chimeric enzyme were inactive or not phosphorylated. We therefore added Asp at position 404 to mimic the introduction of negative charge that would accompany phosphorylation. Enzyme 404D/H398Q was inactive for reaction [1] or [3], but catalyzed reaction [2] at 35% the wild-type rate. These observations are consistent with the model that attenuation of catalytic activity results from an ionic interaction between the imidazolium cation of His 398 and the carboxylate anion of Asp 404.
Bischoff
K M
KM
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
Rodwell
V W
VW
D000595:Amino Acid Sequence; D000818:Animals; D001105:Archaea / Q000201:enzymology*; D002384:Catalysis; D006224:Cricetinae; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008647:Mesocricetus; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment
D011993:Recombinant Fusion Proteins; D006639:Histidine; D006903:Hydroxymethylglutaryl CoA Reductases