title: His865 is the catalytically important histidyl residue of Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
abstract: Involvement in catalysis of a histidyl residue of Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was suggested by the ability of diethyl pyrocarbonate to abolish catalytic activity, accompanying spectral changes, and reactivation by hydroxylamine. The 7 histidines present in the catalytic domain of the hamster enzyme were changed to glutamine (His474, His487, His634, His751, His860, and His865), lysine (His865), or tyrosine (His868). Overexpression in Escherichia coli yielded six soluble mutant proteins, one insoluble protein (H634Q), and one which was degraded in vivo (H487Q). Following purification to homogeneity, mutant enzymes H474Q, H751Q, H860Q, and H868Y had essentially wild-type catalytic activity, while mutant enzymes H865K and H865Q had less than 0.6% wild-type activity. The low activity of mutant enzymes H865K and H865Q is unlikely to reflect altered structural integrity since both chromatographed on affinity supports like wild-type enzyme and had Km values for (S)-HMG-CoA (31 and 16 microM) and for NADPH (60 and 24 microM) close to those for wild-type enzyme (31 and 52 microM for (S)-HMG-CoA and NADPH, respectively). His865 of hamster HMG-CoA reductase, the histidine of the consensus Leu-Val-Xaa-Ser-His-Met-Xaa-Xaa-Asn-Arg-Ser motif and the only histidine conserved among the catalytic domains of all HMG-CoA reductases, thus appears to be a general acid/base functional in catalysis.
authors: Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
authors: B G
authors: BG
authors: Darnay
authors: V W
authors: VW
authors: Rodwell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D017124:Conserved Sequence; D006224:Cricetinae; D004247:DNA; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics / Q000378:metabolism*; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007700:Kinetics; D008647:Mesocricetus; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
chemical_list: D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D006639:Histidine; D004247:DNA; D006903:Hydroxymethylglutaryl CoA Reductases; D004047:Diethyl Pyrocarbonate