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.
Highlights
The prototype of the family, Glc-6-Pase-␣, is a 357-amino acid, nine-transmembrane domain, endoplasmic reticulum (ER)-associated protein [10, 11], which is expressed primarily in the liver, kidney, and intestine [12, 13]
Hydrolysis of glucose 6-phosphate (Glc-6-P) to glucose and phosphate via a covalent phosphohistidine-Glc-6-Pase-␣ intermediate was first proposed by Nordlie and Lygre [23] based on pH kinetic studies of Glc6-Pase-␣ catalysis, which was confirmed by the identification of an enzyme-bound 32P-labeled histidine after incubating rat liver microsomes with [32P]Glc-6-P (24 –26)
In an effort to understand the role of this ubiquitous Glc-6Pase activity, we have undertaken a study of the active site of Glc-6-Pase- and confirmed that it is similar to Glc-6-Pase-␣
Summary
Vol 279, No 13, Issue of March 26, pp. 12479 –12483, 2004 Printed in U.S.A. Histidine 167 Is the Phosphate Acceptor in Glucose-6-phosphatase- Forming a Phosphohistidine Enzyme Intermediate during Catalysis*. During glucose 6-phosphate (Glc-6-P) hydrolysis, Glc-6-Pase-␣, a nine-transmembrane domain protein, forms a covalently bound phosphoryl enzyme intermediate through His176, which lies on the lumenal side of the ER membrane. Critical residues for Glc-6-Pase-␣ catalysis in the active site motif include: Arg, which donates hydrogen ions to the phosphate and stabilizes the transition state; His119, which provides a proton to liberate the glucose moiety; and His176, which undertakes a nucleophilic attack on the phosphate to form a covalently bound phosphoryl enzyme intermediate [21, 27]. Sequence alignments predict that His167 is the residue that forms a covalent bond with phosphate to create a phosphorylGlc-6-Pase- intermediate, whereas Arg and His114 are the hydrogen donors These alignments suggest that Glc-6Pase- contains nine putative transmembrane helices and that Arg, His114, and His167 lie inside the ER lumen. Viously considered to be the only ones capable of contributing to interprandial glucose homeostasis [13]
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