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• W2068964169 abstract "To know the structural properties responsible for the enzymic activity of the 50-kDa C-terminal half of type II hexokinase (HKII-C) derived from rat hepatoma cell line AH130, we constructed cDNAs of HKII-C and its recombinants in which restricted regions containing highly conserved sequences, referred to as regions 2 and 3, were replaced by the corresponding regions of glucokinase. The binding domains of ATP and glucose were proposed to exist in these regions, respectively. Then, the HKII-C and chimera HKII-Cs were overexpressed in Escherichia coli BL21(DE3)pLysS. They all exhibited hexokinase activity, and their activities were inhibited by glucose-6-phosphate (Glc-6-P) competitively for ATP and uncompetitively for glucose. The replacement of region 2 of HKII-C by the corresponding region of glucokinase increased the affinity for glucose and decreased the affinity for Glc-6-P, but it did not significantly affect the affinity for ATP. In contrast, the replacement of region 3 did not cause an appreciable change in hexokinase activity. These findings suggest that region 2 is associated with the binding of ATP and Glc-6-P, and that the latter binding site is located close to the ATP binding site. In addition, region 2 was suggested to be directly related with the binding of glucose and other hexoses. To know the structural properties responsible for the enzymic activity of the 50-kDa C-terminal half of type II hexokinase (HKII-C) derived from rat hepatoma cell line AH130, we constructed cDNAs of HKII-C and its recombinants in which restricted regions containing highly conserved sequences, referred to as regions 2 and 3, were replaced by the corresponding regions of glucokinase. The binding domains of ATP and glucose were proposed to exist in these regions, respectively. Then, the HKII-C and chimera HKII-Cs were overexpressed in Escherichia coli BL21(DE3)pLysS. They all exhibited hexokinase activity, and their activities were inhibited by glucose-6-phosphate (Glc-6-P) competitively for ATP and uncompetitively for glucose. The replacement of region 2 of HKII-C by the corresponding region of glucokinase increased the affinity for glucose and decreased the affinity for Glc-6-P, but it did not significantly affect the affinity for ATP. In contrast, the replacement of region 3 did not cause an appreciable change in hexokinase activity. These findings suggest that region 2 is associated with the binding of ATP and Glc-6-P, and that the latter binding site is located close to the ATP binding site. In addition, region 2 was suggested to be directly related with the binding of glucose and other hexoses. Hexokinase (HK, 1The abbreviations used are: HKhexokinaseHKII-CC-terminal half of type II HKGKglucokinaseKm(ATP)Km for ATPKm(Glc)Km for glucoseKi(ATP)Ki of glucose 6-phosphate with ATPKi(Glc)Ki of glucose 6-phosphate with glucoseGlc-6-Pglucose 6-phosphateVmax(Glc)Vmax for glucoseVmax(ATP)Vmax for ATPIPTGisopropyl-1-thio-β-D-galactopyranosidePCRpolymerase chain reactionLB mediumLuria-Bertani mediumPAGEpolyacrylamide gel electrophoresisHPLChigh performance liquid chromatographyDTTdithiothreitolPK-LDHpyruvate kinase-lactate dehydrogenasev(Glc)initial phosphorylation velocity with glucosev(Fru)initial phosphorylation velocity with fructosev(Man)initial phosphorylation velocity with mannosercorrelation coefficient. ATP:D-hexose 6-phosphotransferase, EC) catalyzes the first step of sequential hexose metabolism in cells (1Wilson J.E. Beitner R. Regulation of Carbohydrate Metabolism. CRC Press Inc., Boca Raton, FL1984: 45Google Scholar), and its activity is known to be abnormally high in tumor cells (2Weinhouse S. Cancer Res. 1972; 32: 2007-2016Google Scholar, 3Bustamante E. Morris H.P. Pedersen P.L. J. Biol. Chem. 1981; 256: 8699-8704Google Scholar, 4Arora K.K. Pedersen P.L. J. Biol. Chem. 1988; 263: 17422-17428Google Scholar, 5Arora K.K. Fanciulli M. Pedersen P.L. J. Biol. Chem. 1990; 265: 6481-6488Google Scholar). Four isozymes of HK in mammalian tissues are known to date. In general, the type I isozyme is expressed in the brain and kidney (6Grossbard L. Schimke R.T. J. Biol. Chem. 1966; 241: 3546-3560Google Scholar), type II in muscle and adipocytes (6Grossbard L. Schimke R.T. J. Biol. Chem. 1966; 241: 3546-3560Google Scholar), type III in cell nuclei (7Preller A. Wilson J.E. Arch. Biochem. Biophys. 1992; 294: 482-492Google Scholar), and type IV in the liver and pancreas (8Ureta T. Comp. Biochem. Physiol. 1982; 71: 549-555Google Scholar). We recently found by Northern blot analysis that type II HK is specifically expressed in tumor cell lines of rat AH130 (9Shinohara Y. Ichihara J. Terada H. FEBS Lett. 1991; 291: 55-57Google Scholar) and human HepG2 (10Shinohara Y. Yamamoto K. Kogure K. Ichihara J. Terada H. Cancer Lett. 1994; 82: 27-32Google Scholar). Furthermore, it is reported that a large amount of type II isozyme exists on the mitochondrial membrane of Morris hepatoma 3924A (11Rempel A. Bannasch P. Mayer D. Biochem. J. 1994; 303: 269-274Google Scholar) and rat hepatoma AS-30D cells (12Mathupala S.P. Rempel A. Pedersen P.L. J. Biol. Chem. 1995; 270: 16918-16925Google Scholar). hexokinase C-terminal half of type II HK glucokinase Km for ATP Km for glucose Ki of glucose 6-phosphate with ATP Ki of glucose 6-phosphate with glucose glucose 6-phosphate Vmax for glucose Vmax for ATP isopropyl-1-thio-β-D-galactopyranoside polymerase chain reaction Luria-Bertani medium polyacrylamide gel electrophoresis high performance liquid chromatography dithiothreitol pyruvate kinase-lactate dehydrogenase initial phosphorylation velocity with glucose initial phosphorylation velocity with fructose initial phosphorylation velocity with mannose correlation coefficient. All of these HK isozymes catalyze the phosphorylation of D-hexoses utilizing MgATP, and undergo feedback inhibition by the phosphorylated products. However, the size and function of the type IV HK isozyme, known as glucokinase (GK), differ from those of other mammalian isozymes. Its molecular mass is about 50 kDa, whereas those of the other isozymes are about 100 kDa, and it mainly phosphorylates glucose under physiological conditions, but its affinity for glucose is very low, its Km value for glucose being about 100 times greater than the other isozymes (1Wilson J.E. Beitner R. Regulation of Carbohydrate Metabolism. CRC Press Inc., Boca Raton, FL1984: 45Google Scholar, 8Ureta T. Comp. Biochem. Physiol. 1982; 71: 549-555Google Scholar). In addition, the type IV isozyme is insensitive to physiological concentrations of the product Glc-6-P (1Wilson J.E. Beitner R. Regulation of Carbohydrate Metabolism. CRC Press Inc., Boca Raton, FL1984: 45Google Scholar, 8Ureta T. Comp. Biochem. Physiol. 1982; 71: 549-555Google Scholar). In this paper, we refer to the type IV isozyme as GK. Analysis of cDNAs encoding mammalian type I-III isozymes showed that they consist of a tandem arrangement of two highly homologous polypeptides, the amino acid sequence of which are very similar to those of the 50-kDa yeast HK and GK (13Schwab D.A. Wilson J.E. J. Biol. Chem. 1988; 263: 3220-3224Google Scholar, 14Schwab D.A. Wilson J.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2563-2567Google Scholar, 15Andreone T.L. Printz R.L. Pilkins S.J. Magnuson M.A. Granner D.K. J. Biol. Chem. 1989; 264: 363-369Google Scholar, 16Schwab D.A. Wilson J.E. Arch. Biochem. Biophys. 1991; 285: 365-370Google Scholar, 17Thelen A.P. Wilson J.E. Arch. Biochem. Biophys. 1991; 286: 645-651Google Scholar). Therefore, it has been thought that the 100-kDa HK is derived from an ancestral protein similar to the 50-kDa yeast HK or GK by gene duplication and fusion (13Schwab D.A. Wilson J.E. J. Biol. Chem. 1988; 263: 3220-3224Google Scholar, 14Schwab D.A. Wilson J.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2563-2567Google Scholar, 15Andreone T.L. Printz R.L. Pilkins S.J. Magnuson M.A. Granner D.K. J. Biol. Chem. 1989; 264: 363-369Google Scholar, 16Schwab D.A. Wilson J.E. Arch. Biochem. Biophys. 1991; 285: 365-370Google Scholar, 17Thelen A.P. Wilson J.E. Arch. Biochem. Biophys. 1991; 286: 645-651Google Scholar, 18Nishi S. Seino S. Bell G.I. Biochem. Biophys. Res. Commun. 1988; 157: 937-943Google Scholar, 19White T.K. Wilson J.E. Arch. Biochem. Biophys. 1989; 274: 375-393Google Scholar, 20White T.K. Wilson J.E. Arch. Biochem. Biophys. 1990; 277: 26-34Google Scholar, 21Magnani M. Bianchi M. Casabianca A. Stocchi V. Daniele A. Altruda F. Ferrone M. Silengo L. Biochem. J. 1992; 285: 193-199Google Scholar). Recently, Printz et al. (22Printz R.L. Koch S. Potter L.R. O'Doherty R.M. Tiesinga J.J. Moritz S. Granner D.K. J. Biol. Chem. 1993; 268: 5209-5219Google Scholar) and we (23Kogure K. Shinohara Y. Terada H. J. Biol. Chem. 1993; 268: 8422-8424Google Scholar) found that the structure of genomic DNA of rat type II HK consists essentially of a duplication of the genomic DNA of rat GK with the same intron/exon structures as the latter. The promoter sequence of the type II HK gene was also determined by us (24Ichihara J. Shinohara Y. Kogure K. Terada H. Biochim. Biophys. Acta. 1995; 1260: 365-368Google Scholar) and Mathupala et al. (12Mathupala S.P. Rempel A. Pedersen P.L. J. Biol. Chem. 1995; 270: 16918-16925Google Scholar). Despite extensive studies, it is still unknown why the enzymatic properties of type I-III HKs differ from that of GK. For understanding the molecular mechanisms of enzyme activity of HKs, the enzyme functions of the two homologous peptide chains of type I HK have received much attention. The function of the 50-kDa C-terminal half of type I HK is well characterized, being directly related with catalytic function and inhibition by Glc-6-P, whereas the role of the 50-kDa N-terminal half is not clear (18Nishi S. Seino S. Bell G.I. Biochem. Biophys. Res. Commun. 1988; 157: 937-943Google Scholar, 19White T.K. Wilson J.E. Arch. Biochem. Biophys. 1989; 274: 375-393Google Scholar, 20White T.K. Wilson J.E. Arch. Biochem. Biophys. 1990; 277: 26-34Google Scholar, 25Arora K.K. Filburn C.R. Pedersen P.L. J. Biol. Chem. 1991; 266: 5359-5362Google Scholar, 26Arora K.K. Filburn C.R. Pedersen P.L. J. Biol. Chem. 1993; 268: 18259-18266Google Scholar, 27Tsai H.J. Wilson J.E. Arch. Biochem. Biophys. 1995; 316: 206-214Google Scholar). It is possible that the N-terminal half, which lacks enzyme activity, is related with regulatory functions (18Nishi S. Seino S. Bell G.I. Biochem. Biophys. Res. Commun. 1988; 157: 937-943Google Scholar, 19White T.K. Wilson J.E. Arch. Biochem. Biophys. 1989; 274: 375-393Google Scholar, 20White T.K. Wilson J.E. Arch. Biochem. Biophys. 1990; 277: 26-34Google Scholar, 27Tsai H.J. Wilson J.E. Arch. Biochem. Biophys. 1995; 316: 206-214Google Scholar), or it may act as an anchor for the binding of HK to the outer mitochondrial membrane, to which most HKs are bound in brain and tumor cells (3Bustamante E. Morris H.P. Pedersen P.L. J. Biol. Chem. 1981; 256: 8699-8704Google Scholar, 4Arora K.K. Pedersen P.L. J. Biol. Chem. 1988; 263: 17422-17428Google Scholar, 11Rempel A. Bannasch P. Mayer D. Biochem. J. 1994; 303: 269-274Google Scholar, 12Mathupala S.P. Rempel A. Pedersen P.L. J. Biol. Chem. 1995; 270: 16918-16925Google Scholar, 28Polakis P.G. Wilson J.E. Arch. Biochem. Biophys. 1985; 236: 328-337Google Scholar, 29Xie G. Wilson J.E. Arch. Biochem. Biophys. 1988; 267: 803-810Google Scholar, 30Smith A.D. Wilson J.E. Arch. Biochem. Biophys. 1991; 287: 359-366Google Scholar). As little is known about the structural basis of the enzymatic activity of type II HK, which is responsible for the high HK activity of tumor cells, we studied the enzymatic property of the C-terminal half of type II HK, referred to as HKII-C. To this end, we constructed a cDNA encoding HKII-C and cDNAs encoding chimera HKII-Cs, consisting of combinations of various regions of cDNAs encoding HKII-C and GK, and overexpressed them in an Escherichia coli strain. We then purified HKII-C and chimera HKII-Cs and examined their catalytic activities under various conditions. DNA restriction endonucleases and Taq DNA polymerase were obtained from Takara Shuzo (Kyoto, Japan). [α-32P]dCTP was purchased from Amersham (Backinghamshire, United Kingdom). The expression plasmid vector pET-3d and its host bacterium BL21(DE3)pLysS were obtained from Novagen (Madison, WI). Other materials and reagents were of the highest grade commercially available. Four cDNA fragments encoding the C-terminal half (nucleotides 1616-1804, 1805-1953, 1954-2133, and 2134-2960) of the rat type II HK and those of rat GK (nucleotides 362–516 and 517–696) containing restriction sites of endonucleases were prepared by PCR. The template used for PCR of HKII-C was the first strand of cDNA reverse-transcribed from mRNA of AH130 ascites tumor cells prepared as described previously (23Kogure K. Shinohara Y. Terada H. J. Biol. Chem. 1993; 268: 8422-8424Google Scholar). The template for amplification of GK cDNA fragments was prepared from rat liver mRNA in the same manner. PCR was carried out with 0.45 µg of the template DNA and 100 pmol of primers specific to rat type II HK or rat GK containing DNA endonuclease sites in a solution of 1.6 mM dNTP, 2.5 units of Taq DNA polymerase, 1.5 mM MgCl2, 50 mM KCl, and 10 mM Tris-HCl buffer (pH 8.3). The primers for PCR were designed to contain the endonuclease restriction sites without altering the amino acid sequences, except for the replacement of Leu by Met at the N-terminal residue of the rat HKII-C as the start codon for translation. These primers are shown schematically in Table I. The template DNA and primers were heated at 94°C for 3 min and then subjected to a chain reaction of 30 cycles of heating at 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min, with final extension at 72°C for 10 min.Table INucleotide sequences of the PCR primers for amplification of cDNA fragments encoding rat HKII-C and rat GKHKII-CGKH1Downstream5′-CAGAAGAAGTCTCTNcoIUpstream5′-TTTCCAAGATCCAAKpnIH2G2Downstream5′-TTGGAAACTTCCGG5′-GACCTGGGAAACTTKpnIKpnIUpstream5′-CATGTAGAAGTCCG5′-GATGCTTGAAGTCAXhoIXhoIH3G3Downstream5′-CGGACTTCTACATG5′-TGACTTCAAGCATCXhoIXhoIUpstream5′-TCCAGGTCCTCTCG5′-CCATCTCCCCTCTCEcoRIEcoRIH4Downstream5′-CGAGAGGACCTGGAEcoRIUpstream5′-GCCCCGTCCCAAGCBamHI Open table in a new tab The cDNA fragments obtained by PCR were subcloned into pUC19 vector, and their nucleotide sequences were determined by the dideoxynucleotide chain termination method (31Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5436-5467Google Scholar) using primers complementary to the vector. The cDNA fragments of HKII-C were ligated into the endonuclease sites of each fragment for construction of a complete cDNA encoding rat HKII-C. For use in the pET expression system (32Lange A.J. Xu L.Z. Van Poelwijk F. Lin K. Granner D.K. Pilkis S.J. Biochem. J. 1991; 277: 159-163Google Scholar), this constructed cDNA was subcloned into expression vector pET-3d. This subcloned plasmid is referred to as pET-HKII-C. Then, cDNAs encoding various chimeric HKII-Cs were constructed based on the cDNAs encoding HKII-C and GK. The constructed plasmids encoding HKII-C and chimeric HKII-Cs were transfected into E. coli strain BL21(DE3)pLysS cells in LB medium containing ampicillin (0.1 mg/ml) and chloramphenicol (34 µg/ml). This E. coli strain is reported to be very useful for the expression of proteins (32Lange A.J. Xu L.Z. Van Poelwijk F. Lin K. Granner D.K. Pilkis S.J. Biochem. J. 1991; 277: 159-163Google Scholar). The transformed cells were cultured in LB medium containing ampicillin and chloramphenicol overnight at 37°C, diluted 100-fold with the same medium, and incubated further at 37°C for 3 h. They were then shaken for 30 min at 22°C, the protein expression inducer IPTG was added at the final concentration of 0.4 mM, and the cell suspension was incubated at 22°C for 6 h. Transformed cells in 1 liter of culture medium were collected by centrifugation at 5,000 × g for 5 min at 4°C, and the pellet was resuspended in 40 ml of 10 mM sodium phosphate buffer (pH 7.5) containing 1 mM EDTA, 1 mM DTT, 10 mM glucose, and 0.5 mM phenylmethylsulfonyl fluoride. The suspension was then frozen and thawed three times, and after the addition of MgSO4 to a final concentration of 50 mM, insoluble materials were removed by centrifugation at 10,000 × g for 1 h at 4°C. The resultant supernatant was used as bacterial extract. The extract was fractionated with (NH4)2SO4. The precipitate with 30–60% saturation of (NH4)2SO4 was dissolved in 10 mM sodium phosphate buffer (pH 7.5) containing 1 mM EDTA, 1 mM DTT, and 10 mM glucose. The protein sample was subjected to gel filtration through a column of Superdex 200 (26 × 600 mm; Pharmacia, Uppsala, Sweden), eluted with 10 mM sodium phosphate buffer (pH 7.5) containing 1 mM EDTA, 1 mM DTT, and 10 mM glucose at a flow rate of 2.5 ml/min, and eluates were collected at 1-min intervals. Fractions containing HK activity were pooled and subjected to ion-exchange HPLC on a column of Resource Q (6.4 × 30 mm, Pharmacia) with a linear gradient of 0–300 mM NaCl containing 10 mM sodium phosphate buffer (pH 7.5), 1 mM EDTA, 1 mM DTT, and 10 mM glucose for 60 min at a flow rate of 1 ml/min monitored by absorbance at 280 nm. The fractions were collected at 1-min intervals, and their HK activities were determined. The chromatography was repeated, and the fractions having HK activity were stored at −20°C after glycerol was added to a final concentration of 50%. The HK activities of HKII-C and chimeric HKII-Cs after storage for at least 1 month were the same as those determined just after purification. By these procedures, HKII-C and all the chimeric HKII-Cs were purified about 10-fold giving more than 90% purity on SDS-PAGE, and their activities were recovered to 10–20% of those in the bacterial extracts. These samples were subjected to gel filtration on a HiTrap desalting column (5 ml, Pharmacia) eluted with 10 mM sodium phosphate buffer (pH 7.5) containing 1 mM EDTA and 1 mM DTT just before determination of enzymatic parameters. The endogenous E. coli HK, with activity of about 0.5-1.0% of those of HKII-C and chimeric HKII-Cs in bacterial extracts, was separated from HKII-C and chimeric HKII-Cs during ion-exchange HPLC. The protein concentration was determined according to the method of Bradford (33Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar) using bovine serum albumin as the standard. The purified HKII-C described above was subjected to reversed-phase HPLC on a TSK gel phenyl-5PWRP column (4.6 × 75 mm, Tosoh, Tokyo, Japan) with a linear gradient of 20–60% acetonitrile containing 0.05% trifluoroacetic acid for 40 min at a flow rate of 1.0 ml/min. The elution profile, monitored by absorbance at 210 nm, showed almost a single peak due to HKII-C, which was eluted at 45% acetonitrile. Then, the amino acid sequence analysis of HKII-C in the eluate was performed in a Shimadzu PSQ-1 gas-phase sequenator, and the resultant phenylthiohydantoin derivatives of amino acids were identified with an on-line HPLC system equipped with a TSK gel PTH Pak (2 × 250 mm, Tosoh) as reported previously (34Majima E. Koike H. Hong Y.-M. Shinohara Y. Terada H. J. Biol. Chem. 1993; 268: 22181-22187Google Scholar). The HK activity was determined from the decrease in absorbance at 340 nm due to conversion of NADH to NAD+ coupled with lactate formation from pyruvate in the presence of phosphoenolpyruvate, lactate dehydrogenase, and pyruvate kinase with various concentrations of either glucose in the presence of 5 mM ATP, or ATP in the presence of 5 mM glucose. The reaction was started by the addition of HKII-C or chimeric HKII-Cs to the medium containing 50 mM Tris-HCl buffer (pH 8.0), 15 mM MgCl2, 100 mM KCl, 0.25 mM NADH, 1 mM phosphoenolpyruvate, 10 units/ml pyruvate kinase, and 10 units/ml lactate dehydrogenase (35Storer A.C. Cornish-Bowden A. Biochem. J. 1976; 159: 7-14Google Scholar). This method is referred to as the PK-LDH method. From the absorbance changes, the values of Km(Glc), Km(ATP), Vmax(Glc) and Vmax(ATP) were determined. The inhibitory effect of Glc-6-P was determined in terms of Ki(Glc) and Ki(ATP). The initial phosphorylation velocity (v) with other hexoses, such as mannose and fructose, was determined by the PK-LDH method. In addition, the enzymatic activities were also determined from the absorbance change at 340 nm due to NADPH formation coupled with 6-phosphogluconolactone formation from Glc-6-P catalyzed by Glc-6-P dehydrogenase in the presence of glucose and ATP (36Bustamante E. Pedersen P.L. Biochemistry. 1980; 19: 4972-4977Google Scholar). For this, HKII-C or chimeric HKII-Cs were added to solutions of 50 mM Tris-HCl buffer (pH 7.6), 15 mM MgCl2, 1 mM NADP+, 0.4 unit/ml Glc-6-P dehydrogenase, and various concentrations of glucose with 5 mM ATP, or ATP with 5 mM glucose. This procedure is referred to as the glucose-6-phosphate dehydrogenase method. Although the Km values determined by the PK-LDH method without added Glc-6-P might be affected by the reaction product Glc-6-P, the values were essentially the same as those determined by the glucose-6-phosphate dehydrogenase method. Putative binding domains of ATP and glucose, and the amino acid residues related with glucose binding of type I HK, GK, and 50-kDa yeast HK have been proposed (5Arora K.K. Fanciulli M. Pedersen P.L. J. Biol. Chem. 1990; 265: 6481-6488Google Scholar, 15Andreone T.L. Printz R.L. Pilkins S.J. Magnuson M.A. Granner D.K. J. Biol. Chem. 1989; 264: 363-369Google Scholar, 37Bennett W.S. Steitz T.A. J. Mol. Biol. 1980; 140: 211-230Google Scholar, 38Arora K.K. Pedersen P.L. Arch. Biochem. Biophys. 1993; 304: 515-518Google Scholar). In the C-terminal half of type I HK, the amino acid residues between Asp532 and Lys558 and those between Met596 and Phe623 are proposed to be binding domains of ATP and glucose, respectively, and Asp657, Glu708, and Glu742 are thought to interact with the hydroxyl group of glucose (5Arora K.K. Fanciulli M. Pedersen P.L. J. Biol. Chem. 1990; 265: 6481-6488Google Scholar, 38Arora K.K. Pedersen P.L. Arch. Biochem. Biophys. 1993; 304: 515-518Google Scholar). As these amino acid residues are well conserved in the C-terminal half of type II HK (HKII-C) as well as GK (cf. Fig. 1), it is reasonable to assume that these domains are associated with the binding of ATP and glucose in HKII-C. To determine the structural basis of the differences in enzymatic activity between HKII-C and GK, we studied the role of these domains in HKII-C by replacement of the regions containing these binding domains by the corresponding regions of GK. Genomic DNA of type II HK consists of a tandem repeat of the GK-like gene and has 18 exons, the 10th exon being formed by linkage of the last exon (10th exon referred to as N10) of the N-side of the gene with the 2nd exon (referred to as C2) of the C-side of the gene (22Printz R.L. Koch S. Potter L.R. O'Doherty R.M. Tiesinga J.J. Moritz S. Granner D.K. J. Biol. Chem. 1993; 268: 5209-5219Google Scholar, 23Kogure K. Shinohara Y. Terada H. J. Biol. Chem. 1993; 268: 8422-8424Google Scholar), as shown in Fig. 2. We constructed a cDNA encoding HKII-C starting from Leu474, amino acid residue 5 in C2. This N-terminal residue was replaced by Met to construct a start codon for translation. Our cDNA for HKII-C consisted of nucleotides 1616-2960 of type II HK, numbered according to Thelen and Wilson (17Thelen A.P. Wilson J.E. Arch. Biochem. Biophys. 1991; 286: 645-651Google Scholar). Sites for the endonucleases KpnI, XhoI, and EcoRI were constructed at nucleotides 1805, 1954, and 2134, respectively, by suitable replacements of these nucleotides by other nucleotides without alteration of the amino acid residues. The cDNA fragments with these nuclease sites were prepared by PCR using the oligonucleotide primers shown in Table I, and are referred to as H1 (corresponding to nucleotides 1616-1804 of type II HK), H2 (nucleotides 1805-1953), H3 (nucleotides 1954-2133), and H4 (nucleotides 2134-2960) in order from the upstream region. Similarly, cDNA fragments of GK containing these endonuclease sites were prepared by PCR and are referred to as G2 (corresponding to nucleotides 362–516 of the cDNA of GK) and G3 (nucleotides 517–696). The nucleotides of GK are numbered as reported by Andreone et al. (15Andreone T.L. Printz R.L. Pilkins S.J. Magnuson M.A. Granner D.K. J. Biol. Chem. 1989; 264: 363-369Google Scholar). From the nucleotide sequences of the cDNAs of type II HK and GK, H2 and H3, respectively, are regarded to correspond to G2 and G3. Most of the putative binding domain of ATP (Thr536-Lys558) and the whole putative binding domain of glucose (Leu596-Phe623) proposed for type I HK and GK (5Arora K.K. Fanciulli M. Pedersen P.L. J. Biol. Chem. 1990; 265: 6481-6488Google Scholar, 15Andreone T.L. Printz R.L. Pilkins S.J. Magnuson M.A. Granner D.K. J. Biol. Chem. 1989; 264: 363-369Google Scholar, 37Bennett W.S. Steitz T.A. J. Mol. Biol. 1980; 140: 211-230Google Scholar, and 38; cf. Fig. 1) exist, respectively, in H2 and H3, and in G2 and G3. Based on these structural features, we constructed cDNAs of three chimera HKII-Cs H1G2H3H4, H1H2G3H4, and H1G2G3H4, in which H2, H3, and H2 plus H3 were replaced, respectively, by G2, G3, and G2 plus G3, as shown in Fig. 2. First, we tried to overexpress the rat HKII-C in E. coli BL21(DE3)pLysS transformed by pET-HKII-C, the plasmid containing cDNA encoding HKII-C. As shown in Fig. 3 (lanes A and B), the protein expression inducer IPTG induced production of only the 50-kDa protein in the bacteria transformed by pET-HKII-C, and the bacterial extract showed the HK activity (specific activity, 21 µmol/min/mg protein). Both of the HK activities of the extract of untransformed bacteria and that of the bacteria transformed by the plasmid vector pET-3d lacking cDNA of HKII-C were 0.1 µmol/min/mg protein, but the 50-kDa protein was not detected in these samples on SDS-PAGE under the present experimental conditions (data not shown). For purification of the 50-kDa protein thus overexpressed, the proteins in bacterial extract were fractionated with (NH4)2SO4, and the precipitate with 30–60% saturation of (NH4)2SO4 was solubilized (lane C). The protein sample thus obtained was subjected to gel filtration on a column of Superdex 200, and the fractions having HK activity (lane D) were subjected to ion-exchange HPLC on a column of Resource Q eluted with a linear gradient of NaCl. The elution profile is shown in Fig. 4. The endogenous HK, the activity of which was detected at fraction 14, apart from the fractions of HKII-C, was removed in this step. The ion-exchange HPLC was repeated with fractions having HK activity, and a resultant protein having HK activity of 133 µmol/min/mg protein gave almost a single 50-kDa band on SDS-PAGE (Fig. 3, lane E).Fig. 4Purification by ion-exchange HPLC of overexpressed HKII-C. The fractions having HK activity in gel filtration of the precipitate with (NH4)2SO4 of bacterial extract (cf. legends for Fig. 3) were subjected to ion-exchange HPLC on a column of Resource Q with a linear gradient of NaCl at a flow rate of 1 ml/min. The absorbances of the eluates were monitored at 280 nm, shown by a continuous line, and their HK activities, shown by closed circles, were determined by the glucose-6-phosphate dehydrogenase method. Ion-exchange HPLC was repeated with fractions 31–33, and the protein sample in fractions having HK activity on SDS-PAGE (cf. Fig. 3, lane E) was used for examination of HK activities.View Large Image Figure ViewerDownload (PPT) N-terminal sequence analysis of this purified protein showed the sequence of Met-Glu-Ser-Leu-Lys-Leu-Ser-His-Glu-Gln-Leu-Leu-Glu-Val-Lys-Arg-Arg, which is identical to that of the designed HKII-C, indicating that this 50-kDa protein is HKII-C having HK activity. By similar procedures, the chimeric HKII-Cs H1G2H3H4, H1H2G3H4, and H1G2G3H4 were overexpressed (Fig. 5A) and purified (Fig. 5B). The specific activities of these chimeric HKII-Cs in the bacterial extracts were 10–20 µmol/min/mg protein, and those in the purified samples were about 10-fold higher. We measured the initial phosphorylation velocity (v) of the purified HKII-C as a function of the concentration (C) of the substrates ATP with 5 mM glucose and glucose with 5 mM ATP, either in the absence or presence of various concentrations of Glc-6-P. The linear relationships between 1/v and 1/C for ATP and glucose, analyzed by the least-squares method, are shown in Fig. 6, Fig. 7, respectively. The intercepts on the y axis of all the straight lines were the same, with different slopes for the substrate ATP in the presence of 5 mM glucose, whereas the slopes were parallel with different intercepts for glucose in the presence of 5 mM ATP, showing that HKII-C activities for ATP and for glucose were competitively and uncompetitively inhibited by Glc-6-P, respectively, as observed with HK isozymes (1Wilson J.E. Beitner R. Regulation of Carbohydrate Metabolism. CRC Press Inc., Boca Raton, FL1984: 45Google Scholar, 6Grossbard L. Schimke R.T. J. Biol. Chem. 1966; 241: 3546-3560Google Scholar, 26Arora K.K. Filburn C.R. Pedersen P.L. J. Biol. Chem. 1993; 268: 18259-18266Google Scholar). We determined the values of Km(Glc) and Km(ATP), Vmax(Glc) and Vmax(ATP), and Ki(Glc) and Ki(ATP) by Glc-6-P for each run in at least three separate experiments, and their mean values are shown in Table II. The values of Km and Ki of HKII-C were found to be slightly greater than those of type II HK isozyme overexpressed in E. coli BL21(DE3)pLysS (22Printz R.L. Koch S. Potter L.R. O'Doherty R.M. Tiesinga J.J. Moritz S. Granner D.K. J. Biol. Chem. 1993; 268: 5209-5219Google Scholar); Km(Glc), Km(ATP) and Ki" @default.
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• W2068964169 title "Alteration of Enzyme Function of the Type II Hexokinase C-terminal Half on Replacements of Restricted Regions by Corresponding Regions of Glucokinase" @default.
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