Histidine 167 Is the Phosphate Acceptor in Glucose-6-phosphatase-β Forming a Phosphohistidine Enzyme Intermediate during Catalysis

The glucose-6-phosphatase (Glc-6-Pase) family comprises two active endoplasmic reticulum (ER)-associated isozymes: the liver/kidney/intestine Glc-6-Pase-α and the ubiquitous Glc-6-Pase-β. Both share similar kinetic properties. Sequence alignments predict the two proteins are structurally similar. Du...

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Bibliographic Details
Published in:The Journal of biological chemistry 2004-03, Vol.279 (13), p.12479-12483
Main Authors: Ghosh, Abhijit, Shieh, Jeng-Jer, Pan, Chi-Jiunn, Chou, Janice Yang
Format: Article
Language:English
Subjects:
Adenoviridae - genetics
Amino Acid Sequence
Animals
Binding Sites
Blotting, Western
Catalysis
Cell Membrane - metabolism
COS Cells
Cyanogen Bromide - pharmacology
Endoplasmic Reticulum - metabolism
Epitopes - chemistry
Glucose - metabolism
Glucose-6-Phosphatase - chemistry
Histidine - analogs & derivatives
Histidine - chemistry
Humans
Hydrolysis
Isoelectric Focusing
Kinetics
Microsomes - metabolism
Molecular Sequence Data
Mutation
Precipitin Tests
Protein Conformation
Protein Isoforms
Protein Structure, Tertiary
Sequence Homology, Amino Acid
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Summary:The glucose-6-phosphatase (Glc-6-Pase) family comprises two active endoplasmic reticulum (ER)-associated isozymes: the liver/kidney/intestine Glc-6-Pase-α and the ubiquitous Glc-6-Pase-β. 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-α, a nine-transmembrane domain protein, forms a covalently bound phosphoryl enzyme intermediate through His176, which lies on the lumenal side of the ER membrane. We showed that Glc-6-Pase-β 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-β active site mutants R79A, H114A, and H167A. Using [32P]Glc-6-P coupled with cyanogen bromide mapping, we demonstrated that the phosphate acceptor in Glc-6-Pase-β is His167 and that it lies inside the ER lumen with the active site residues, Arg79 and His114. Therefore Glc-6-Pase-α and Glc-6-Pase-β share a similar active site structure, topology, and mechanism of action.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M313271200