Phosphorylation of Ser871 impairs the function of His865 of Syrian hamster 3-hydroxy-3-methylglutaryl-CoA reductase.
The attenuation of catalytic activity that accompanies phosphorylation of Ser871 of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) reflects primarily the introduction of negative charge (Omkumar, R. V., Darnay, B. G., and Rodwell, V. W. (1994) J. Biol. Chem. 269, 6810-6814). To investigate how a negative charge at position 871 attenuates activity, we phosphorylated wild-type and mutant HMG-CoA reductases and assayed reduction of the putative intermediate mevaldehyde to mevalonate. We observed attenuated activity when the phosphorylated wild-type enzyme was assayed in the presence or absence of coenzyme A, but not when assayed in the presence of desthio-CoA. These observations recall the behavior of mutant enzyme H865Q, for which coenzyme A inhibits, whereas desthio-CoA stimulates mevaldehyde reduction (Frimpong, K. F., and Rodwell, V. W. (1994) J. Biol. Chem. 269, 11478-11483). Catalysis of mevaldehyde reduction by mutant enzyme H865Q was unaffected by phosphorylation. By contrast, mutant enzymes H860Q and H868Y, in which nearby, but noncatalytic, histidines had been mutated, exhibited wild-type behavior upon phosphorylation. We conclude that the introduction of negative charge at position 871 impairs the function of His865, presumably by a specific electrostatic interaction. We propose a novel mechanism by which phosphorylation regulates activity. Phosphorylation of the terminal serine of the consensus AGxLV(K/R)SHMxxNRS motif of eukaryotic HMG-CoA reductases attenuates activity by impairing the ability of the catalytic histidine to protonate the CoAS- anion formed during the reductive deacylation of HMG-CoA to mevaldehyde.
Omkumar
R V
RV
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
Rodwell
V W
VW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003065:Coenzyme A / Q000378:metabolism; D016384:Consensus Sequence; D006224:Cricetinae; D006639:Histidine; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008647:Mesocricetus; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010944:Plants / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine; D013997:Time Factors
D011994:Recombinant Proteins; D012694:Serine; D006639:Histidine; D006903:Hydroxymethylglutaryl CoA Reductases; D003065:Coenzyme A