Histidine 167 Is the Phosphate Acceptor in Glucose-6-phosphatase-β Forming a Phosphohistidine Enzyme Intermediate during Catalysis (2004)

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. 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. The glucose-6-phosphatase (Glc-6-Pase) 1The abbreviations used are: Glc-6-Pase, glucose-6-phosphatase; Glc-6-P, glucose 6-phosphate; ER, endoplasmic reticulum; UGRP, ubiquitously expressed Glc-6-Pase-related protein; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. family is composed of three proteins: Glc-6-Pase-α (1Lei K.-J. Shelly L.L. Pan C.-J. Sidbury J.B. Chou J.Y. Science. 1993; 262: 580-583Crossref PubMed Scopus (320) Google Scholar, 2Shelly L.L. Lei K.-J. Pan C.-J. Sakata S.F. Ruppert S. Schutz G. Chou J.Y. J. Biol. Chem. 1993; 268: 21482-21485Abstract Full Text PDF PubMed Google Scholar, 3Lange A.J. Argaud D. El-Maghrabi M.R. Pan W. Maitra S.R. Pilkis S.J. Biochem. Biophys. Res. Commun. 1994; 201: 302-309Crossref PubMed Scopus (116) Google Scholar, 4Kishnani P.S. Bao Y. Wu J.Y. Brix A.E. Lin J.L. Chen Y.T. Biochem. Mol. Med. 1997; 61: 168-177Crossref PubMed Scopus (47) Google Scholar), Glc-6-Pase-β (5Martin C.C. Oeser J.K. Svitek C.A. Hunter S.I. Hutton J.C. O'Brien R.M. J. Mol. Endocrinol. 2002; 29: 205-222Crossref PubMed Scopus (74) Google Scholar, 6Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 7Guionie O. Clottes E. Stafford K. Burchell A. FEBS Lett. 2003; 551: 159-164Crossref PubMed Scopus (61) Google Scholar) (previously known as UGRP (ubiquitously expressed Glc-6-Pase related protein)), and islet-specific Glc-6-Pase-related protein (8Arden S.D. Zahn T. Steegers S. Webb S. Bergman B. O'Brien R.M. Hutton J.C. Diabetes. 1999; 48: 531-542Crossref PubMed Scopus (94) Google Scholar, 9Martin C.C. Bischof L.J. Bergman B. Hornbuckle L.A. Hilliker C. Frigeri C. Wahl D. Svitek C.A. Wong R. Goldman J.K. Oeser J.K. Lepretre F. Froguel P. O'Brien R.M. Hutton J.C. J. Biol. Chem. 2001; 276: 25197-25207Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Whereas Glc-6-Pase-α and Glc-6-Pase-β are functional phosphohydrolases, the islet-specific Glc-6-Pase-related protein lacks enzymatic activity. The prototype of the family, Glc-6-Pase-α, is a 357-amino acid, nine-transmembrane domain, endoplasmic reticulum (ER)-associated protein (10Pan C.-J. Lei K.-J. Annabi B. Hemrika W. Chou J.Y. J. Biol. Chem. 1998; 273: 6144-6148Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 11Pan C.-J. Lei K.-J. Chou J.Y. J. Biol. Chem. 1998; 273: 21658-21662Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), which is expressed primarily in the liver, kidney, and intestine (12Pan C.-J. Lei K.-J. Chen H. Ward J.M. Chou J.Y. Arch. Biochem. Biophys. 1998; 358: 17-24Crossref PubMed Scopus (60) Google Scholar, 13Nordlie R.C. Sukalski K.A. Martonosi A.N. The Enzymes of Biological Membranes. 2nd Ed. Plenum Press, New York1985: 349-398Crossref Google Scholar). Glc-6-Pase-α catalyzes the hydrolysis of glucose 6-phosphate (Glc-6-P) to glucose in the terminal step of gluconeogenesis and glycogenolysis (13Nordlie R.C. Sukalski K.A. Martonosi A.N. The Enzymes of Biological Membranes. 2nd Ed. Plenum Press, New York1985: 349-398Crossref Google Scholar). Between meals, the resulting release of glucose to the blood maintains glucose homeostasis. Naturally occurring loss of function mutations in Glc-6-Pase-α cause glycogen storage disease type Ia, a disorder that is characterized by loss of blood glucose homeostasis and disorders of glycogen and lipid metabolism (reviewed in Refs. 14Chou J.Y. Matern D. Mansfield B.C. Chen Y.-T. Curr. Mol. Med. 2002; 2: 121-143Crossref PubMed Scopus (227) Google Scholar and 15Chen Y.-T. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Childs B. Kinzler K.W. Vogelstein B. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill Inc., New York2001: 1521-1551Google Scholar). Glc-6-Pase-β is a ubiquitously expressed, 346-amino acid membrane protein that shares a 36% sequence identity to Glc-6-Pase-α (5Martin C.C. Oeser J.K. Svitek C.A. Hunter S.I. Hutton J.C. O'Brien R.M. J. Mol. Endocrinol. 2002; 29: 205-222Crossref PubMed Scopus (74) Google Scholar, 6Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 7Guionie O. Clottes E. Stafford K. Burchell A. FEBS Lett. 2003; 551: 159-164Crossref PubMed Scopus (61) Google Scholar). Despite the absence of any apparent ER retention motif, Glc-6-Pase-β is also localized in the ER membrane (6Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), although its orientation in the membrane is not known. The subcellular localization of the Glc-6-Pase-β active site is not known, although it is assumed to be similar to Glc-6-Pase-α. Both Glc-6-Pase-β and Glc-6-Pase-α couple with the Glc-6-P transporter to form an active Glc-6-Pase complex, and both share similar kinetic properties with respect to Glc-6-P hydrolysis (6Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The active site of Glc-6-Pase-α was originally identified by the presence of a conserved phosphatase signature motif found in lipid phosphatases, acid phosphatases, and vanadium haloperoxidases (16Stukey J. Carman G.M. Protein Sci. 1997; 6: 469-472Crossref PubMed Scopus (222) Google Scholar, 17Hemrika W. Wever R. FEBS Lett. 1997; 409: 317-319Crossref PubMed Scopus (46) Google Scholar). This motif was shown to contribute to the active site of a vanadium chloroperoxidase by both x-ray crystal structure analysis (18Hemrika W. Renirie R. Dekker H.L. Barnett P. Wever R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2145-2149Crossref PubMed Scopus (172) Google Scholar) and mutational studies (19Hemrika W. Renirie R. Macedo-Ribeiro S. Messerschmidt A. Wever R. J. Biol. Chem. 1999; 274: 23820-23827Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 20Renirie R. Hemrika W. Wever R. J. Biol. Chem. 2000; 275: 11650-11657Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) and has since been shown to have a similar role in Glc-6-Pase-α (21Ghosh A. Shieh J.-J. Pan C.-J. Sun M.-S. Chou J.Y. J. Biol. Chem. 2002; 277: 32837-32842Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 22Shieh J.-J. Terizioglu M. Hiraiwa H. Marsh J. Pan C.-J. Chen L.-Y. Chou J.Y. J. Biol. Chem. 2002; 277: 5047-5053Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Hydrolysis of Glc-6-P to glucose and phosphate via a covalent phosphohistidine-Glc-6-Pase-α intermediate was first proposed by Nordlie and Lygre (23Nordlie R.C. Lygre D.G. J. Biol. Chem. 1966; 241: 3136-3141Abstract Full Text PDF PubMed Google Scholar) based on pH kinetic studies of Glc-6-Pase-α catalysis, which was confirmed by the identification of an enzyme-bound 32P-labeled histidine after incubating rat liver microsomes with [32P]Glc-6-P (24Parvin R. Smith R.A. Biochemistry. 1969; 8: 1748-1755Crossref PubMed Scopus (27) Google Scholar, 25Feldman F. Butler L.G. Biochem. Biophys. Res. Commun. 1969; 36: 119-125Crossref PubMed Scopus (38) Google Scholar, 26Feldman F. Butler L.G. Biochim. Biophys. Acta. 1972; 268: 698-710Crossref PubMed Scopus (37) Google Scholar). Critical residues for Glc-6-Pase-α catalysis in the active site motif include: Arg83, 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 (21Ghosh A. Shieh J.-J. Pan C.-J. Sun M.-S. Chou J.Y. J. Biol. Chem. 2002; 277: 32837-32842Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 27Chou J.Y. Curr. Mol. Med. 2001; 1: 25-44Crossref PubMed Scopus (41) Google Scholar). In Glc-6-Pase-α, all of these residues lie together on the lumenal side of the ER membrane (10Pan C.-J. Lei K.-J. Annabi B. Hemrika W. Chou J.Y. J. Biol. Chem. 1998; 273: 6144-6148Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 11Pan C.-J. Lei K.-J. Chou J.Y. J. Biol. Chem. 1998; 273: 21658-21662Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Given the sequence similarity between Glc-6-Pase-α and Glc-6-Pase-β, a similar active site structure would be anticipated. Sequence alignments predict that His167 is the residue that forms a covalent bond with phosphate to create a phosphoryl-Glc-6-Pase-β intermediate, whereas Arg79 and His114 are the hydrogen donors. These alignments also suggest that Glc-6-Pase-β contains nine putative transmembrane helices and that Arg79, His114, and His167 lie inside the ER lumen. In an effort to understand the role of this ubiquitous Glc-6-Pase activity, we have undertaken a study of the active site of Glc-6-Pase-β and confirmed that it is similar to Glc-6-Pase-α. This information can now be used to design experiments that disrupt the activity of Glc-6-Pase-β and investigate the biological importance of a Glc-6-Pase activity that is not limited in expression to the liver, kidney, and intestine, the organs previously considered to be the only ones capable of contributing to interprandial glucose homeostasis (13Nordlie R.C. Sukalski K.A. Martonosi A.N. The Enzymes of Biological Membranes. 2nd Ed. Plenum Press, New York1985: 349-398Crossref Google Scholar). Construction of Glc-6-Pase-β Mutants—Mutants were constructed using the same general strategy used for Glc-6-Pase-α (22Shieh J.-J. Terizioglu M. Hiraiwa H. Marsh J. Pan C.-J. Chen L.-Y. Chou J.Y. J. Biol. Chem. 2002; 277: 5047-5053Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Briefly, the human Glc-6-Pase-β cDNA was engineered to remove the internal SfiI site (6Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and then used as a template for PCR-based N-terminal epitope tagging using either the FLAG (DYKDDDDK) or His10 epitopes. The resulting PCR products were cloned directionally into the pSVL vector to generate pSVL-Glc-6-Pase-β-5FLAG and pSVL-Glc-6-Pase-β-His10. The human pSVL-Glc-6-Pase-β-3FLAG construct has been described by Shieh, et al. (6Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). For the protease protection assays, Glc-6-Pase-β constructs incorporating two cleavable factor Xa tetrapeptide (IEGR) (28Wilkinson B.M. Critchley A.J. Stirling C.J. J. Biol. Chem. 1996; 271: 25590-25597Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) recognition motifs were constructed by PCR and cloned into the pSVL vector. To locate the factor Xa tetrapeptide recognition motifs between the N-terminal FLAG epitope and the Glc-6-Pase-β coding sequence, a 5′ primer containing an ATG initiation codon followed by the 24-bp FLAG coding sequence, two fXa recognition sequences (back to back) (ATCGAGGGTAGAATCGAGGGTAGA), and nucleotides 217–237 of Glc-6-Pase-β (GenBank™ accession number XM_045901) was used. The 3′ primer represented nucleotides 1237–1257 of human Glc-6-Pase-β. The resulting construct was named Glc-6-Pase-β-5fXaFLAG. To locate the factor Xa tetrapeptide recognition motifs between the Glc-6-Pase-β coding sequence and the FLAG the 5′ primer represented nucleotides 217–237 of human Glc-6-Pase-β, whereas the 3′ primer human Glc-6-Pase-β nucleotides 1237–1257 followed by the 24-bp fXa recognition sequence, the 24-bp FLAG coding sequence, and a This construct was named constructs were by Construction of Glc-6-Pase-β containing was by the described by et al. S. M. T. Y. J. 1997; PubMed Google Scholar). The was and to with of The and were described previously (6Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). and in were with of or in the pSVL vector as described previously (10Pan C.-J. Lei K.-J. Annabi B. Hemrika W. Chou J.Y. J. Biol. Chem. 1998; 273: 6144-6148Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). for in of and pH were with for The or were then with for followed by and to The or were with of factor Xa protease for in a containing pH and The factor Xa was then by the of to a of The or factor were to with and for to the The were in and used for The with to disrupt the followed by or factor Xa were used as For were by through a and first with a the FLAG epitope and then with The was with the containing the of the Glc-6-P was glucose and by and by a as described previously (21Ghosh A. Shieh J.-J. Pan C.-J. Sun M.-S. Chou J.Y. J. Biol. Chem. 2002; 277: 32837-32842Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). were with a a of of after for as described previously (6Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). For active site microsomes were for in a containing pH and glucose and then by the of of [32P]Glc-6-P to a of for the was by the of an of acid followed by on for The proteins were then by for and with of pH and with of The was then in a containing pH and the protease The enzyme intermediate was by using a in with protein and identified by and of 32P-labeled phosphoryl proteins were by acid as described The was with of pH and with of and in of pH containing the protease The proteins were bound to a with A. The was to the and the bound protein was with of pH and for The resulting proteins were by through a and the intermediate by containing the protein was with by incubating the in for and used for cyanogen bromide of and of the Glc-6-Pase-β by and intermediate was in of a containing of cyanogen of acid B.M. H.L. in Protein Science. Inc., New Scholar, E. J. Biochem. Biophys. 1997; PubMed Scopus Google Scholar) and in the the products were using in by through a and by containing the 32P-labeled phosphoryl was with and The resulting 32P-labeled phosphoryl-Glc-6-Pase-β was then characterized by through a and by through a pH of Glc-6-Pase-β is in the ER membrane by nine-transmembrane (10Pan C.-J. Lei K.-J. Annabi B. Hemrika W. Chou J.Y. J. Biol. Chem. 1998; 273: 6144-6148Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 11Pan C.-J. Lei K.-J. Chou J.Y. J. Biol. Chem. 1998; 273: 21658-21662Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) with the in the ER lumen and the in the analysis of the Glc-6-Pase-β acid sequence using the K. W. Biol. Chem. 1993; Scholar) that Glc-6-Pase-β is also in the ER by nine putative transmembrane helices this is the and of the protein be on of the ER membrane. To the subcellular localization of the of Glc-6-Pase-β, we protease protection using and Glc-6-Pase-β microsomes with or were with and the protein were by analysis with an In microsomes were to However, the is not a In experiments using microsomes by both and FLAG are by of and human Glc-6-Pase-β. microsomes or were to by in the absence or presence of as described microsomes or were to by factor Xa protease in the absence or presence of as described and Glc-6-Pase-β were with an of are in we confirmed the of Glc-6-Pase-β by a site into factor Xa protease recognition motif (28Wilkinson B.M. Critchley A.J. Stirling C.J. J. Biol. Chem. 1996; 271: 25590-25597Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) was between the Glc-6-Pase-β coding and the FLAG to the and with the factor Xa of microsomes a protein, whereas of microsomes to loss of the FLAG epitope of the have shown previously that during catalysis, in Glc-6-Pase-α as a phosphate acceptor a covalently bound phosphoryl enzyme intermediate (21Ghosh A. Shieh J.-J. Pan C.-J. Sun M.-S. Chou J.Y. J. Biol. Chem. 2002; 277: 32837-32842Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The kinetic and between Glc-6-Pase-β and Glc-6-Pase-α suggest that a phosphoryl enzyme intermediate is also during Glc-6-Pase-β or were with and the resulting intermediate was by and by and a enzyme intermediate was identified in not in R79A, H114A, or active site His167 the in on the nine domain of Glc-6-Pase-β the active site residues, Arg79, His114, and are to lie inside the lumen of the Sequence His167 is the phosphate acceptor in Glc-6-Pase-β. Glc-6-Pase-β contains nine residues and which can be by cyanogen bromide B.M. H.L. in Protein Science. Inc., New Scholar, E. J. Biochem. Biophys. 1997; PubMed Scopus Google Scholar). the His167 is to lie a with a of To the intermediate, we a Glc-6-Pase-β construct a His10 the were with and the intermediate was by and by through a The intermediate was then by cyanogen and the products were through a with an apparent of and with apparent of and were identified The to the of the of To that the of the the was the and by and The 32P-labeled as a through a with an The that in Glc-6-Pase-β, His167 is the acid that covalently the phosphoryl during Glc-6-Pase-β glucose homeostasis between on gluconeogenesis and In the terminal step to both Glc-6-P is to glucose by the Glc-6-Pase complex, which is composed of two coupled the Glc-6-P transporter and a Glc-6-P (reviewed in 14Chou J.Y. Matern D. Mansfield B.C. Chen Y.-T. Curr. Mol. Med. 2002; 2: 121-143Crossref PubMed Scopus (227) Google Scholar). only functional Glc-6-Pase been This previously Glc-6-Pase, is expressed in the liver/kidney/intestine with its role in glucose homeostasis (reviewed in 13Nordlie R.C. Sukalski K.A. Martonosi A.N. The Enzymes of Biological Membranes. 2nd Ed. Plenum Press, New York1985: 349-398Crossref Google Scholar). a ubiquitously expressed Glc-6-Pase-related protein, which was previously UGRP and to be (5Martin C.C. Oeser J.K. Svitek C.A. Hunter S.I. Hutton J.C. O'Brien R.M. J. Mol. Endocrinol. 2002; 29: 205-222Crossref PubMed Scopus (74) Google Scholar), was demonstrated to be active (6Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 7Guionie O. Clottes E. Stafford K. Burchell A. FEBS Lett. 2003; 551: 159-164Crossref PubMed Scopus (61) Google Scholar). a the proteins have been Glc-6-Pase-α and Glc-6-Pase-β Glc-6-Pase-α is the kinetic activity (6Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and ER localization (6Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) of Glc-6-Pase-β is known. a first step to the biological of Glc-6-Pase-β, we have to it is structurally similar to Glc-6-Pase-α, a protein in the ER by nine-transmembrane (10Pan C.-J. Lei K.-J. Annabi B. Hemrika W. Chou J.Y. J. Biol. Chem. 1998; 273: 6144-6148Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 11Pan C.-J. Lei K.-J. Chou J.Y. J. Biol. Chem. 1998; 273: 21658-21662Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The active site residues in Glc-6-Pase-α have been characterized (21Ghosh A. Shieh J.-J. Pan C.-J. Sun M.-S. Chou J.Y. J. Biol. Chem. 2002; 277: 32837-32842Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 22Shieh J.-J. Terizioglu M. Hiraiwa H. Marsh J. Pan C.-J. Chen L.-Y. Chou J.Y. J. Biol. Chem. 2002; 277: 5047-5053Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) and shown to lie into the lumen of the During catalysis, Glc-6-Pase-α forms a phosphoryl enzyme intermediate (23Nordlie R.C. Lygre D.G. J. Biol. Chem. 1966; 241: 3136-3141Abstract Full Text PDF PubMed Google Scholar, R. Smith R.A. Biochemistry. 1969; 8: 1748-1755Crossref PubMed Scopus (27) Google Scholar, 25Feldman F. Butler L.G. Biochem. Biophys. Res. Commun. 1969; 36: 119-125Crossref PubMed Scopus (38) Google Scholar, 26Feldman F. Butler L.G. Biochim. Biophys. Acta. 1972; 268: 698-710Crossref PubMed Scopus (37) Google Scholar) by the of a covalent bond between the phosphoryl of Glc-6-P and in Glc-6-Pase-α (21Ghosh A. Shieh J.-J. Pan C.-J. Sun M.-S. Chou J.Y. J. Biol. Chem. 2002; 277: 32837-32842Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Glc-6-Pase-β lacks any apparent ER transmembrane protein retention motif, studies have shown that it is localized in the ER (6Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). of the of Glc-6-Pase-β using the K. W. Biol. Chem. 1993; Scholar) that Glc-6-Pase-β is in the membrane by nine Using Glc-6-Pase-β constructs and a protease protection by limited we found that the of Glc-6-Pase-β is to the that the of the protein a that is with a nine-transmembrane domain of in the Glc-6-Pase-β by the between residues and would the to that Given the the FLAG this that the FLAG epitope lies on a the first residues of Glc-6-Pase-β. Sequence to the nine-transmembrane domain of Glc-6-Pase-α (10Pan C.-J. Lei K.-J. Annabi B. Hemrika W. Chou J.Y. J. Biol. Chem. 1998; 273: 6144-6148Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 11Pan C.-J. Lei K.-J. Chou J.Y. J. Biol. Chem. 1998; 273: 21658-21662Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) to the first site that lies in the putative are between residues and in Glc-6-Pase-β, by this the first site is to lie the first transmembrane the site lies the between and transmembrane the two lie the transmembrane and the site lies the of the first the protease protection were using constructs engineered to a protease site the were with a and lumenal Glc-6-Pase-β has been shown to have an number of transmembrane and are with the nine-transmembrane both by analysis K. W. Biol. Chem. 1993; Scholar) and sequence to Glc-6-Pase-α. In an we showed that Glc-6-Pase-β with the Glc-6-P transporter to form an active Glc-6-Pase (6Shieh J.-J. Pan C.-J. Mansfield B.C. Chou J.Y. J. Biol. Chem. 2003; 278: 47098-47103Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). This is also with the nine-transmembrane domain that the active site residues Arg79, His114, and His167 on the lumenal side of the membrane. The kinetic and similarity between Glc-6-Pase-α and Glc-6-Pase-β catalysis that the mechanism of of the active site is also similar. In the hydrolysis of Glc-6-P, Glc-6-Pase-α forms a covalently bound intermediate between and the phosphate (21Ghosh A. Shieh J.-J. Pan C.-J. Sun M.-S. Chou J.Y. J. Biol. Chem. 2002; 277: 32837-32842Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The residue in Glc-6-Pase-β is Using 32P-labeled Glc-6-P we the intermediate microsomes Glc-6-Pase-β. the intermediate is not detectable in microsomes the Glc-6-Pase-β active site mutants R79A, H114A, or H167A. To His167 as the phosphate we the intermediate by and of the intermediate with cyanogen bromide is to create The His167 is to lie on a with a of the intermediate a of a which is with His167 as the phosphate In of and were also bound to the cyanogen bromide whereas the were and The with that The is with the containing the is with the containing and the is with the containing that His167 as the the enzyme intermediate during Glc-6-Pase-β In we showed that the ubiquitous Glc-6-Pase-β is in the ER by nine-transmembrane helices with its active site inside the the liver/kidney/intestine Glc-6-Pase-α. The similarity between Glc-6-Pase-β and Glc-6-Pase-α also to the of the active site residues Arg79, His114, and His167 and the of a covalently bound phosphoryl enzyme intermediate during We are now in a to study the of of Glc-6-Pase-β and its importance to blood glucose homeostasis and metabolism in We C. Mansfield for of the