title: Histidine 167 is the phosphate acceptor in glucose-6-phosphatase-beta forming a phosphohistidine enzyme intermediate during catalysis.
abstract: The glucose-6-phosphatase (Glc-6-Pase) family comprises two active endoplasmic reticulum (ER)-associated isozymes: the liver/kidney/intestine Glc-6-Pase-alpha and the ubiquitous Glc-6-Pase-beta. Both share similar kinetic properties. Sequence alignments predict the two proteins are structurally similar. During glucose 6-phosphate (Glc-6-P) hydrolysis, Glc-6-Pase-alpha, a nine-transmembrane domain protein, forms a covalently bound phosphoryl enzyme intermediate through His(176), which lies on the lumenal side of the ER membrane. We showed that Glc-6-Pase-beta is also a nine-transmembrane domain protein that forms a covalently bound phosphoryl enzyme intermediate during Glc-6-P hydrolysis. However, the intermediate was not detectable in Glc-6-Pase-beta active site mutants R79A, H114A, and H167A. Using [(32)P]Glc-6-P coupled with cyanogen bromide mapping, we demonstrated that the phosphate acceptor in Glc-6-Pase-beta is His(167) and that it lies inside the ER lumen with the active site residues, Arg(79) and His(114). Therefore Glc-6-Pase-alpha and Glc-6-Pase-beta share a similar active site structure, topology, and mechanism of action.
authors.affiliation: Section on Cellular Differentiation, Heritable Disorders Branch, National Institute of Child Health & Human Development, National Institutes of Health, Building 10 Rm. 9S241, Bethesda, MD 20892, USA.
authors.forename: Abhijit
authors.initials: A
authors.lastname: Ghosh
authors.forename: Jeng-Jer
authors.initials: JJ
authors.lastname: Shieh
authors.forename: Chi-Jiunn
authors.initials: CJ
authors.lastname: Pan
authors.forename: Janice Yang
authors.initials: JY
authors.lastname: Chou
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D003488:Cyanogen Bromide / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D005947:Glucose / Q000378:metabolism; D005952:Glucose-6-Phosphatase / Q000737:chemistry*; D006639:Histidine / Q000031:analogs & derivatives* / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D007525:Isoelectric Focusing; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011233:Precipitin Tests; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
chemical_list: D000939:Epitopes; D020033:Protein Isoforms; D006639:Histidine; D005952:Glucose-6-Phosphatase; D005947:Glucose; D003488:Cyanogen Bromide; C005436:phosphohistidine