Modulation of Syrian hamster 3-hydroxy-3-methylglutaryl-CoA reductase activity by phosphorylation. Role of serine 871.
Attenuation of Syrian hamster 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) activity by in vitro phosphorylation was studied using AMP-activated protein kinase and wild-type and mutant forms of HMG-CoA reductase. The only residue of the wild-type enzyme phosphorylated was Ser871. Substrates protected against kinase-mediated attenuation of activity, consistent with substrate-induced conformational changes at the C-terminal region. Although close to the catalytic histidine His865, Ser871 appears to play no direct role in catalysis or substrate recognition. Mutant enzymes S871A, S871H, S871N, and S871Q exhibited from 62-106% of wild-type activity and had wild-type Km values for HMG-CoA and NADPH. Replacement of Ser871 by aspartate or glutamate, but not by glutamine, asparagine, histidine, or tyrosine, severely attenuated activity. Attenuation of catalytic activity that accompanies phosphorylation thus appears to result primarily from the introduction of negative charge, not merely steric hindrance. Other than the wild-type enzyme, only mutant enzyme S871T was phosphorylated, and phosphorylation was accompanied by attenuation of activity. The AMP-activated kinase thus can also phosphorylate threonyl residues.
Omkumar
R V
RV
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
Darnay
B G
BG
Rodwell
V W
VW
D055372:AMP-Activated Protein Kinases; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006903:Hydroxymethylglutaryl CoA Reductases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008647:Mesocricetus; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010766:Phosphorylation; D017354:Point Mutation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012694:Serine; D000255:Adenosine Triphosphate; D004247:DNA; D006903:Hydroxymethylglutaryl CoA Reductases; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases