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Amino Acid Changes within Antenna Helix Are Responsible for Different Regulatory Preferences of Human Glutamate Dehydrogenase Isozymes

2007; Elsevier BV; Volume: 282; Issue: 27 Linguagem: Inglês

10.1074/jbc.m703018200

1083-351X

Myung‐Min Choi, Eun‐A Kim, Seung-Ju Yang, Soo Young Choi, Sung‐Woo Cho, Jae‐Wan Huh,

Neuroscience and Neuropharmacology Research

Human glutamate dehydrogenase (hGDH) exists in hGDH1 (housekeeping isozyme) and in hGDH2 (nerve-specific isozyme), which differ markedly in their allosteric regulation. Because they differ in only 16 of their 505 amino acids, the regulatory preferences must arise from amino acid residues that are not common between hGDH1 and hGDH2. To our knowledge none of the mutagenesis studies on the hGDH isozymes to date have identified the amino acid residues fully responsible for the different regulatory preferences between hGDH1 and hGDH2. In this study we constructed hGDH1(hGDH2390–448)hGDH1 (amino acid segment 390–448 of hGDH1 replaced by the corresponding hGDH2 segment) and hGDH2(hGDH1390–448)hGDH2 (amino acid segment 390–448 of hGDH2 replaced by the corresponding hGDH1 segment) by swapping the corresponding amino acid segments in hGDH1 and hGDH2. The chimeric enzymes by reciprocal swapping resulted in double mutations in amino acid sequences at 415 and 443 residues that are not common between hGDH1 and hGDH2 and are located in the C-terminal 48-residue "antenna" helix, which is thought to be part of the regulatory domain of mammalian GDHs. Functional analyses revealed that the doubly mutated chimeric enzymes almost completely acquired most of the different regulatory preferences between hGDH1 and hGDH2 for electrophoretic mobility, heat-stability, ADP activation, palmitoyl-CoA inhibition, and l-leucine activation, except for GTP inhibition. Our results indicate that substitutions of the residues in the antenna region may be important evolutionary changes that led to the adaptation of hGDH2 to the unique metabolic needs of the nerve tissue. Human glutamate dehydrogenase (hGDH) exists in hGDH1 (housekeeping isozyme) and in hGDH2 (nerve-specific isozyme), which differ markedly in their allosteric regulation. Because they differ in only 16 of their 505 amino acids, the regulatory preferences must arise from amino acid residues that are not common between hGDH1 and hGDH2. To our knowledge none of the mutagenesis studies on the hGDH isozymes to date have identified the amino acid residues fully responsible for the different regulatory preferences between hGDH1 and hGDH2. In this study we constructed hGDH1(hGDH2390–448)hGDH1 (amino acid segment 390–448 of hGDH1 replaced by the corresponding hGDH2 segment) and hGDH2(hGDH1390–448)hGDH2 (amino acid segment 390–448 of hGDH2 replaced by the corresponding hGDH1 segment) by swapping the corresponding amino acid segments in hGDH1 and hGDH2. The chimeric enzymes by reciprocal swapping resulted in double mutations in amino acid sequences at 415 and 443 residues that are not common between hGDH1 and hGDH2 and are located in the C-terminal 48-residue "antenna" helix, which is thought to be part of the regulatory domain of mammalian GDHs. Functional analyses revealed that the doubly mutated chimeric enzymes almost completely acquired most of the different regulatory preferences between hGDH1 and hGDH2 for electrophoretic mobility, heat-stability, ADP activation, palmitoyl-CoA inhibition, and l-leucine activation, except for GTP inhibition. Our results indicate that substitutions of the residues in the antenna region may be important evolutionary changes that led to the adaptation of hGDH2 to the unique metabolic needs of the nerve tissue. Glutamate dehydrogenase (GDH) 3The abbreviations used are: GDH, glutamate dehydrogenase; hGDH, human GDH; HPLC, high performance liquid chromatography. 3The abbreviations used are: GDH, glutamate dehydrogenase; hGDH, human GDH; HPLC, high performance liquid chromatography. is found in all organisms and catalyzes the oxidative deamination of glutamate to 2-oxoglutarate. Although this enzyme does not exhibit allosteric regulation in plants, bacteria, or fungi, its activity is tightly controlled by a number of compounds in mammals (1Yip K.S.P. Stillman T.J. Britton K.L. Artymiuk P.J. Baker P.J. Sedelnikova S.E. Engel P.C. Pasquo A. Chiaraluce R. Consalvi V. Scandurra R. Rice D.W. Structure. 1995; 3: 1147-1158Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 2Knapp S. de Vos W.M. Rice D. Ladenstein R. J. Mol. Biol. 1997; 267: 916-932Crossref PubMed Scopus (133) Google Scholar, 3Zaganas I. Plaitakis A. J. Biol. Chem. 2002; 277: 26422-26428Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 4Yang S.-J. Huh J.-W. Hong H.-N. Kim T.U. Cho S.-W. FEBS Lett. 2004; 562: 59-64Crossref PubMed Scopus (19) Google Scholar, 5Mastorodemos V. Zaganas I. Spanaki C. Bessa M. Plaitakis A. J. Neurosci. Res. 2005; 79: 65-73Crossref PubMed Scopus (62) Google Scholar, 6Kanavouras K. Mastorodemos V. Borompokas N. Spanaki C. Plaitakis A. J. Neurosci. Res. 2007; 85: 1101-1109Crossref PubMed Scopus (12) Google Scholar). All mammalian GDHs are homohexameric, exhibiting 32 symmetry, and the first 200 residues form the core "glutamate binding" domain. On top of this domain is a "NAD binding" domain that rotates about 20° during catalysis (7Smith T.J. Schmidt T. Fang J. Wu J. Siuzdak G. Stanley C.A. J. Mol. Biol. 2002; 318: 765-777Crossref PubMed Scopus (105) Google Scholar, 8Banerjee S. Schmidt T. Fang J. Stanley C.A. Smith T.J. Biochemistry. 2003; 42: 3446-3456Crossref PubMed Scopus (74) Google Scholar, 9Allen A. Kwagh J. Fang J. Stanley C.A. Smith T.J. Biochemistry. 2004; 43: 14431-14443Crossref PubMed Scopus (45) Google Scholar). Unique to the animal structures is a 48-residue antenna that protrudes above this NAD binding domain (7Smith T.J. Schmidt T. Fang J. Wu J. Siuzdak G. Stanley C.A. J. Mol. Biol. 2002; 318: 765-777Crossref PubMed Scopus (105) Google Scholar, 8Banerjee S. Schmidt T. Fang J. Stanley C.A. Smith T.J. Biochemistry. 2003; 42: 3446-3456Crossref PubMed Scopus (74) Google Scholar, 9Allen A. Kwagh J. Fang J. Stanley C.A. Smith T.J. Biochemistry. 2004; 43: 14431-14443Crossref PubMed Scopus (45) Google Scholar). Various roles of GDH have been reported. Partial deficiency of GDH has been reported in some patients with cerebellar degeneration, suggesting that the enzymes are important in brain function (10Hussain M.H. Zannis V.I. Plaitakis A. J. Biol. Chem. 1989; 264: 20730-20735Abstract Full Text PDF PubMed Google Scholar). GDH has shown neuroprotective value in model systems where glutamate reuptake is inhibited (11Gorovitis R. Avidan N. Avisar N. Shaked I. Vardimon L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7024-7029Crossref PubMed Scopus (121) Google Scholar). In addition, it has been reported that inhibition of GDH expression by antisense oligonucleotides was toxic to cultured mesencephalic neurons, with dopaminergic neurons being affected at the early stages of this inhibition (12Plaitakis A. Shashidharan P. J. Neurol. 2000; 247: 25-35Google Scholar). The existence of the hyperinsulinism-hyperammonemia syndrome further highlights the importance of GDH in the regulation of insulin secretion and indicates that GDH has an important role in regulating hepatic ureagenesis (13Stanley C.A. Lieu Y.K. Hus B.Y. Burlina A.B. Greenberg C.R. Hopwood N.J. Perlman K. Rich B.H. Zammarchi E. Ponoz M. N. Engl. J. Med. 1998; 338: 1352-1357Crossref PubMed Scopus (605) Google Scholar, 14Tanizawa Y. Nakai K. Sasaki T. Anno T. Ohta Y. Inoue H. Matsuo K. Koga M. Furukawa S. Oka Y. Diabetes. 2002; 51: 712-717Crossref PubMed Scopus (45) Google Scholar). In the human, GDH exists in a housekeeping isozyme (hGDH1) encoded by the GLUD1 gene and a neural and testicular tissue-specific isozyme (hGDH2) encoded by the GLUD2 gene (15Shashidharan P. Michaelidis T.M. Robakis N.K. Kresovali A. Papamatheakis J. Plaitakis A. J. Biol. Chem. 1994; 269: 16971-16976Abstract Full Text PDF PubMed Google Scholar). Although the two GDH isozymes are highly homologous (showing a 97% amino acid identity), they differ markedly in their regulatory properties such as heat stability and allosteric regulation by ADP, l-leucine, and GTP (3Zaganas I. Plaitakis A. J. Biol. Chem. 2002; 277: 26422-26428Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 4Yang S.-J. Huh J.-W. Hong H.-N. Kim T.U. Cho S.-W. FEBS Lett. 2004; 562: 59-64Crossref PubMed Scopus (19) Google Scholar, 5Mastorodemos V. Zaganas I. Spanaki C. Bessa M. Plaitakis A. J. Neurosci. Res. 2005; 79: 65-73Crossref PubMed Scopus (62) Google Scholar, 6Kanavouras K. Mastorodemos V. Borompokas N. Spanaki C. Plaitakis A. J. Neurosci. Res. 2007; 85: 1101-1109Crossref PubMed Scopus (12) Google Scholar, 16Shashidharan P. Clarke D.D. Ahmed N. Moschonas N. Plaitakis A. J. Neurochem. 1997; 68: 1804-1811Crossref PubMed Scopus (78) Google Scholar, 17Zaganas I. Spanaki C. Karpusas M. Plaitakis A. J. Biol. Chem. 2002; 277: 46552-46558Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Because the hGDH isozymes differ in only 16 of their 505 amino acids, these functional differences must arise from amino acid residues that are not common between hGDH1 and hGDH2. Reciprocally interchanging amino acids that are different between hGDH isozymes within the regulatory domain may reveal the residues important for preference in hGDH isozymes. Recent studies of structure-function relationships using site-directed mutagenesis of hGDH1 at single sites differing from hGDH2 showed that the R443S and the G456A change reproduced some but not all of the properties of hGDH2 (3Zaganas I. Plaitakis A. J. Biol. Chem. 2002; 277: 26422-26428Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 4Yang S.-J. Huh J.-W. Hong H.-N. Kim T.U. Cho S.-W. FEBS Lett. 2004; 562: 59-64Crossref PubMed Scopus (19) Google Scholar, 5Mastorodemos V. Zaganas I. Spanaki C. Bessa M. Plaitakis A. J. Neurosci. Res. 2005; 79: 65-73Crossref PubMed Scopus (62) Google Scholar, 17Zaganas I. Spanaki C. Karpusas M. Plaitakis A. J. Biol. Chem. 2002; 277: 46552-46558Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In addition, several other residues differing between hGDH isozymes also have been examined by many investigators (4Yang S.-J. Huh J.-W. Hong H.-N. Kim T.U. Cho S.-W. FEBS Lett. 2004; 562: 59-64Crossref PubMed Scopus (19) Google Scholar, 17Zaganas I. Spanaki C. Karpusas M. Plaitakis A. J. Biol. Chem. 2002; 277: 46552-46558Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Very recently, Kanavouras et al. (6Kanavouras K. Mastorodemos V. Borompokas N. Spanaki C. Plaitakis A. J. Neurosci. Res. 2007; 85: 1101-1109Crossref PubMed Scopus (12) Google Scholar) created a double hGDH1 mutant differing from hGDH2 and showed that the double-mutated enzyme with R443S and G456A in the same polypeptide chain did not acquire all the characteristics of the wild-type hGDH2. These results suggest that additional amino acid changes should be responsible for the unique properties of the brain-specific hGDH2 isozyme. To our knowledge, none of the single or multiple mutagenesis studies on the hGDH isozymes up to date have identified the amino acid residues fully responsible for the different regulatory properties between the hGDH1 and hGDH2. Our objective is to identify these critical residues by multiple mutagenesis. In this study we selected two such residues, Met-415 and Arg-443, because they are the only two amino acid residues that are different between hGDH1 and hGDH2 within the C-terminal 48-residue antenna region, which is thought to be part of the regulatory domain of mammalian GDH (7Smith T.J. Schmidt T. Fang J. Wu J. Siuzdak G. Stanley C.A. J. Mol. Biol. 2002; 318: 765-777Crossref PubMed Scopus (105) Google Scholar, 8Banerjee S. Schmidt T. Fang J. Stanley C.A. Smith T.J. Biochemistry. 2003; 42: 3446-3456Crossref PubMed Scopus (74) Google Scholar, 9Allen A. Kwagh J. Fang J. Stanley C.A. Smith T.J. Biochemistry. 2004; 43: 14431-14443Crossref PubMed Scopus (45) Google Scholar). Using cassette mutagenesis, we constructed hGDH1(hGDH2390–448)hGDH1 (amino acid segment 390–448 of hGDH1 replaced by the corresponding hGDH2 segment) and hGDH2(hGDH1390–448)hGDH2 (amino acid segment 390–448 of hGDH2 replaced by the corresponding hGDH1 segment) by swapping the corresponding amino acid segments between 390 and 448 regions in hGDH1 and hGDH2. The mutated cDNAs were expressed in Escherichia coli as soluble proteins, and the chimeric forms of hGDH isozymes were purified to homogeneity and studied with respect to their kinetic and regulatory characteristics. The functional and evolutionary implications of these findings are discussed. Materials—ADP, GTP, palmitoyl-CoA, and l-leucine were purchased from Sigma-Aldrich. 2,5-ADP-Sepharose and Resource-Q were purchased from Amersham Biosciences. Restriction enzymes were purchased from New England Biolabs. All other chemicals and solvents were reagent grade or better. E. coli DH5α (18Hanahan D. J. Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8098) Google Scholar) was purchased from Invitrogen and used as the host strain for plasmid-mediated transformations for cassette mutagenesis. E. coli PA340 (thr-1 fhuA2 leuB6 lacY1 supE44 gal-6 gdh-1 hisG1 rfbD1 galP63 Δ(gltB-F)500 rpsL19 malT1 xyl-7 mtl-2 argH1 thi-1; kindly provided by Dr. Mary K.B. Berlyn, E. coli Genetic Stock Center, Yale University, New Haven, CT) lacked both GDH and glutamate synthase activities (19Teller J.K. Smith R.J. McPherson M.J. Engel P.C. Guest J.R. Eur. J. Biochem. 1992; 206: 151-159Crossref PubMed Scopus (109) Google Scholar) and was used to test plasmids for GDH activity. E. coli BL21 (DE3) (20Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4772) Google Scholar) was used for high level expression of the recombinant enzymes. Construction of hGDH1(hGDH2390–448)hGDH1 and hGDH2(hGDH1390–448)hGDH2 Chimeric Enzymes—Using cassette mutagenesis, we created two double mutations, each containing both of two amino substitutions at 415 and 443 sites in the same polypeptide chain. The strategy to construct the chimeric enzymes is shown in Fig. 1. Synthetic hGDH1 and hGDH2 genes used in this study were previously constructed in our laboratory (4Yang S.-J. Huh J.-W. Hong H.-N. Kim T.U. Cho S.-W. FEBS Lett. 2004; 562: 59-64Crossref PubMed Scopus (19) Google Scholar, 21Cho S.-W. Yoon H.-Y. Ahn J.-Y. Lee E.-Y. Lee J. Eur. J. Biochem. 2001; 268: 3205-3213Crossref PubMed Scopus (25) Google Scholar). Using cassette mutagenesis, the construction of hGDH1(hGDH2390–448)hGDH1 (amino acid segment 390–448 of hGDH1 replaced by the corresponding hGDH2 segment) and hGDH2(hGDH1390–448)hGDH2 (amino acid segment 390–448 of hGDH2 replaced by the corresponding hGDH1 segment) was carried out by swapping the corresponding amino acid segments between 390 and 448 regions in hGDH1 and hGDH2. For this construct, the plasmids containing hGDH1 gene (21Cho S.-W. Yoon H.-Y. Ahn J.-Y. Lee E.-Y. Lee J. Eur. J. Biochem. 2001; 268: 3205-3213Crossref PubMed Scopus (25) Google Scholar) and hGDH2 gene (4Yang S.-J. Huh J.-W. Hong H.-N. Kim T.U. Cho S.-W. FEBS Lett. 2004; 562: 59-64Crossref PubMed Scopus (19) Google Scholar) were separately double-digested with AflII and NheI, and the large and small fragments were isolated from the agarose gel. The small and large fragments were then cross-ligated to produce the chimeric constructs hGDH1(hGDH2390–448)GDH1 and hGDH2(hGDH1390–448)GDH2. The successful construction of the chimeric enzymes was confirmed by dideoxy-DNA sequencing analysis. Expression and Purification of Chimeric Enzymes—The chimeric constructs were separately transformed into E. coli/DE3 for overexpression. Fresh overnight cultures of the E. coli/DE3 were used to inoculate 1 liter of LB medium containing 100 μg/ml ampicillin. The cell was grown at 37 °C until the A600 reached 1.0, and then isopropyl 1-thio-β-d-galactopyranoside was added to a final concentration of 1 mm. After isopropyl 1-thio-β-d-galactopyranoside induction, each E. coli/DE3 was grown for an additional 3 h at 37 °C and then harvested by centrifugation. Cell pellets were suspended in 50 ml of 50 mm Tris-HCl, pH 7.2, 1 mm phenylmethylsulfonyl fluoride and lysed with a sonicator. Cellular debris was removed by centrifugation, and the crude extracts were loaded onto an 2,5-ADP-Sepharose column (1.5 × 5 cm) that was equilibrated with buffer A (50 mm Tris-HCl, pH 7.2, and 1 mm β-mercaptoethanol). The column was washed with buffer A until the breakthrough peak of protein had been eluted. The enzyme was then performed using a NaCl step gradient that went from 0 to 300 mm in 30 min at a flow rate of 1 ml/min. The fractions containing GDH activity were pooled, concentrated, and applied to a fast protein liquid chromatograph Resource-Q anion exchange column equilibrated with buffer B (50 mm Tris-HCl, pH 8.0, 0.5 mm EDTA, and 1 mm β-mercaptoethanol). The enzyme was then eluted using a linear gradient made with buffer B in increasing concentrations of NaCl up to 500 mm at 1 ml/min. The purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis using monoclonal antibodies previously produced in our laboratory (22Jang S.H. Kim A.Y. Bahn J.H. Eum W.S. Kim D.W. Park J. Lee K.S. Kang T.-C. Won M.H. Kang J.H. Kwon O.-S. Yoon H.-Y. Lee E.-Y. Cho S.-W. Choi S.Y. Exp. Mol. Med. 2003; 35: 249-256Crossref PubMed Scopus (3) Google Scholar). Protein concentration was determined by the method of Bradford (23Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). Additional N-terminal residues (MIEGR) were removed by treatment with factor Xa (10 μg/1 mg of GDH) and purified by a HPLC gel filtration method as described before (4Yang S.-J. Huh J.-W. Hong H.-N. Kim T.U. Cho S.-W. FEBS Lett. 2004; 562: 59-64Crossref PubMed Scopus (19) Google Scholar, 21Cho S.-W. Yoon H.-Y. Ahn J.-Y. Lee E.-Y. Lee J. Eur. J. Biochem. 2001; 268: 3205-3213Crossref PubMed Scopus (25) Google Scholar). HPLC-purified proteins were subjected to automated amino acid sequencing. Enzyme Assay and Kinetics—Steady state kinetic parameters were determined with the purified proteins unless otherwise indicated. Enzyme assays were performed by monitoring reduced coenzyme absorbance at 340 nm. Because E. coli only has an NADP+-dependent GDH (19Teller J.K. Smith R.J. McPherson M.J. Engel P.C. Guest J.R. Eur. J. Biochem. 1992; 206: 151-159Crossref PubMed Scopus (109) Google Scholar), the enzyme assay was performed with NADH as a coenzyme. All assays were performed in duplicate, and initial velocity data were correlated with a standard assay mixture of 1 ml containing 50 mm triethanolamine, pH 8.0, 100 mm ammonium acetate, 0.1 mm NADH, and 2 mm EDTA, pH 8.0, at 25 °C. Enzyme assay was performed in the presence of 1 mm ADP unless otherwise indicated. The reaction started with the addition of α-ketoglutarate to 2 mm final concentration. The Km values were calculated by linear regression analysis of double-reciprocal plots, and catalytic efficiency was estimated by use of the equation v/[E0] = (kcat/Km)[S] (24Fersht A. Enzyme Structure and Mechanism. W. H. Freeman and Co., New York1985: 98-120Google Scholar). For the heat stability studies, the hGDH isozymes were incubated at 45 °C in the absence of ADP. At various times, aliquots were withdrawn to determine remaining activities by the addition of the standard assay mixture in the presence of 1 mm ADP. Regulation of Chimeric Enzymes—Effects of allosteric regulators on GDH activities were examined by incubating the enzyme with the allosteric effectors at various concentrations in the assay buffer at 25 °C. At intervals after the initiation with the effectors, aliquots were withdrawn for the assay of GDH activity. Regulation of the hGDH isozymes and the chimeric enzymes by ADP or l-leucine was studied by adding ADP (0–1 mm final concentrations) or l-leucine (0–5 mm final concentrations) to the reaction mixture at various concentrations while keeping the other substrates constant. We also explored the effect of ADP and l-leucine used together at various concentrations on hGDH isozymes and chimeric mutants. ADP (0–1 mm) was added to the reaction mixture that contained l-leucine at various concentrations (0–5 mm). GTP inhibition and palmitoyl-CoA inhibition were studied by adding GTP (0–100 μm final concentrations) or palmitoyl-CoA (0–10 μm final concentrations) at various concentrations to the standard reaction mixture in the presence of 1 mm ADP. For the thermal stability studies, the wild-type and chimeric enzymes were incubated in 100 mm potassium phosphate buffer, pH 7.0 at 45 °C in the presence of 1 mm ADP. At various times, aliquots were withdrawn, and the remaining activities were assayed by the addition of the standard assay mixture. Construction, Expression, and Purification of hGDH1-(hGDH2390–448)hGDH1 and hGDH2(hGDH1390–448)hGDH2 Chimeric Enzymes—We have reported construction of synthetic hGDH1 and hGDH2 genes and expression of hGDH1 and hGDH2 isozymes from E. coli as soluble proteins (4Yang S.-J. Huh J.-W. Hong H.-N. Kim T.U. Cho S.-W. FEBS Lett. 2004; 562: 59-64Crossref PubMed Scopus (19) Google Scholar, 21Cho S.-W. Yoon H.-Y. Ahn J.-Y. Lee E.-Y. Lee J. Eur. J. Biochem. 2001; 268: 3205-3213Crossref PubMed Scopus (25) Google Scholar). Using cassette mutagenesis, the construction of hGDH1(hGDH2390–448)hGDH1 (amino acid segment 390–448 of hGDH1 replaced by the corresponding hGDH2 segment) and hGDH2(hGDH1390–448)hGDH2 (amino acid segment 390–448 of hGDH2 replaced by the corresponding hGDH1 segment) was carried out by swapping the corresponding amino acid segments in hGDH1 and hGDH2 (Fig. 1). The chimeric enzymes by reciprocal swapping resulted in double mutations in amino acid sequences at 415 and 443 residues that are not common between hGDH1 and hGDH2. In the wild-type hGDH isozymes, the 415 site is Met in hGDH1 and Leu in hGDH2, and the 443 site is Arg in hGDH1 and Ser in hGDH2. In hGDH1(hGDH2390–448)hGDH1, the 415 site is Leu and the 443 site is Ser, whereas in hGDH2(hGDH1390–448)hGDH2, the 415 site is Met and the 443 site is Arg (Fig. 1). High level expression of the chimeric enzymes was achieved in E. coli strain DE3 upon induction with 1 mm isopropyl-l-d-thiogalactose at 37 °C for 3 h. Analysis of crude cell extracts by Western blot showed that expression levels of hGDH1(hGDH2390–448)hGDH1 and hGDH2(hGDH1390–448)hGDH2 were almost identical to those of wild-type hGDH isozymes (data not shown). The expressed enzymes were purified by ADP-Sepharose column and fast protein liquid chromatograph Resource-Q column. To remove five additional N-terminal residues (MIEGR) that were introduced to create an initiation codon and a factor Xa recognition site, the purified enzymes were treated with factor Xa, purified by an HPLC Protein-Pak 300SW gel filtration column, and subjected to automated Edman degradation. N-terminal sequence analysis of the first eight amino acids was identical with the published sequence of the mature hGDH isozymes (15Shashidharan P. Michaelidis T.M. Robakis N.K. Kresovali A. Papamatheakis J. Plaitakis A. J. Biol. Chem. 1994; 269: 16971-16976Abstract Full Text PDF PubMed Google Scholar, 25Julliard J. Smith E.L. J. Biol. Chem. 1979; 254: 3427-3438Abstract Full Text PDF PubMed Google Scholar). The purified wild-type hGDH2 was estimated to be ∼96% pure by SDS-PAGE (Fig. 2A). Compared with hGDH1, the hGDH2 protein is more basic and shows a slightly lower electrophoretic mobility (Fig. 2A). These differences are consistent with the electrophoretic characteristics of hGDH isozymes as reported by other investigators (15Shashidharan P. Michaelidis T.M. Robakis N.K. Kresovali A. Papamatheakis J. Plaitakis A. J. Biol. Chem. 1994; 269: 16971-16976Abstract Full Text PDF PubMed Google Scholar). Interestingly, when the amino acid segment 390–448 of hGDH1 was replaced by the corresponding hGDH2 segment to make hGDH1(hGDH2390–448)hGDH1, it was found to show the same electrophoretic mobility as hGDH2 (Fig. 2A). Likewise, when the amino acid segment 390–448 from the hGDH1 is spliced onto human GDH2, the electrophoretic mobility of hGDH2(hGDH1390–448)hGDH2 was almost identical to that of hGDH1. These results suggest that the amino acid differences at 415 and 443 sites within the antenna region are responsible for the electrophoretic mobility of the hGDH isozymes. Heat Stability of hGDH1(hGDH2390–448)hGDH1 and hGDH2(hGDH1390–448)hGDH2—The relative resistance of hGDH2 to thermal inactivation was determined by incubation of the enzyme at 45 °C, pH 7.0. Heat inactivation proceeded faster for hGDH2 (half-life = 45 min) than for hGDH1 (half-life = 310 min) (Fig. 2B), supporting the previous observation that hGDH1 is the heat-stable form, and hGDH2 is the heat-labile form of human GDH (4Yang S.-J. Huh J.-W. Hong H.-N. Kim T.U. Cho S.-W. FEBS Lett. 2004; 562: 59-64Crossref PubMed Scopus (19) Google Scholar, 16Shashidharan P. Clarke D.D. Ahmed N. Moschonas N. Plaitakis A. J. Neurochem. 1997; 68: 1804-1811Crossref PubMed Scopus (78) Google Scholar). Under these conditions, the hGDH1(hGDH2390–448)hGDH1 and hGDH2(hGDH1390–448)hGDH2 chimeric enzymes showed the reciprocal changes in thermal stability of hGDH isozymes. hGDH2(hGDH1390–448)hGDH2 abolished the heat lability of hGDH2 and changed the half-life of hGDH2 from 45 to ∼280 min at 45 °C, which is comparable with that of hGDH1 (Fig. 2B). In contrast, hGDH1(hGDH2390–448)hGDH1 abolished the heat stability of hGDH1, and the result obtained with hGDH1(hGDH2390–448)hGDH1 was similar to that observed with hGDH2 (Fig. 2B). Previously, we reported that single replacement of Ser by Arg at hGDH2 position 443 abolished the heat lability of hGDH2 and changed the half-life of hGDH2 to almost the same as that of hGDH1, whereas single mutagenesis at several other sites (L415M, A456G, and H470R) that are different between hGDH1 and hGDH2 did not show any change in thermal stability (5Mastorodemos V. Zaganas I. Spanaki C. Bessa M. Plaitakis A. J. Neurosci. Res. 2005; 79: 65-73Crossref PubMed Scopus (62) Google Scholar). Therefore, the influence of heat stability on hGDH isozymes caused by swapping amino acid segment 390–448 is mainly due to the difference of the amino acid at the 443 site, which is Arg in hGDH1 and Ser in hGDH2. Catalytic Properties of hGDH1(hGDH2390–448)hGDH1 and hGDH2(hGDH1390–448)hGDH2—To evaluate more detailed catalytic properties for the wild-type hGDH isozymes and the chimeric enzymes, the enzyme efficiencies (kcat/Km) for the individual substrates were determined. The catalytic and kinetic properties of hGDH1(hGDH2390–448)hGDH1 and hGDH2(hGDH1390–448)hGDH2 were not altered. The apparent Km values for α-ketoglutarate and NADH were essentially similar for all wild-type and mutant proteins as of 79 ∼ 86 μm and 1.25 ∼ 1.39 mm for NADH and α-ketoglutarate, respectively (Table 1). These results suggest that substitutions at position 415 and 443 site might have no appreciable effect on the affinity of hGDH isozymes for both substrate and coenzyme. However, there was an ∼30% increase in kcat values of wild-type hGDH2 (130 s–1) and hGDH2(hGDH1390–448)hGDH2 (127 s–1) compared with that of the wild-type hGDH1 (104 s–1) and hGDH1(hGDH2390–448)hGDH1 (105 s–1). The increased enzyme efficiency (kcat/Km) of wild-type hGDH2 and hGDH2(hGDH1390–448)hGDH2, therefore, results from the increase in kcat values. Western blot analysis showed that expression levels of hGDH1(hGDH2390–448)hGDH1 and hGDH2(hGDH1390–448)hGDH2 were almost identical to those of wild-type hGDH isozymes (data not shown). In addition, hGDH1(hGDH2390–448)hGDH1 and hGDH2(hGDH1390–448)hGDH2 were purified by the same methods as was the wild-type hGDH isozymes, indicating that no gross conformational change in the chimeric enzymes had occurred. Therefore, the difference in the kcat values between hGDH1 and hGDH2 is probably not due to the amino acid differences at 415 and 443 sites within the antenna region but due to the rest of the amino acids not common between hGDH1 and hGDH2.TABLE 1Kinetic properties of hGDH isozymes and chimeric enzymesEnzymekcatKm-NADHKm-α-KGkcat/Km-NADHkcat/Km-α-KGs-1μmmms-1 μm-1s-1 mm-1hGDH1104811.251.2883.2hGDH2130861.391.5193.5hGDH1(hGDH2390-448)hGDH1105791.281.3382.0hGDH2(hGDH1390-448)hGDH2127801.371.5992.7 Open table in a new tab Inhibition of hGDH1(hGDH2390–448)hGDH1 and hGDH2(hGDH1390–448)hGDH2 by Palmitoyl-CoA—Palmitoyl-CoA induced a concentration-dependent inhibition of hGDH isozymes. Palmitoyl-CoA especially exerted a power inhibitory effect on the hGDH2. In contrast, hGDH1 was less sensitive to palmitoyl-CoA inhibition. When the segment 390–448 from the hGDH2 is spliced onto human GDH1 to make hGDH1(hGDH2390–448)hGDH1, it was found to show almost identical sensitivity to palmitoyl-CoA inhibitory aspects of hGDH2 (Fig. 3A). In contrast, hGDH2(hGDH1390–448)hGDH2 resulted in the resistance to palmitoyl-CoA inhibition that is comparable with that of wild-type hGDH1 (Fig. 3A). These results suggest that the amino acid differences at 415 and 443 sites within the antenna region are responsible for the different sensitivity to inhibition by palmitoyl-CoA of hGDH1 and hGDH2. Activation of hGDH1(hGDH2390–448)hGDH1 and hGDH2(hGDH1390–448)hGDH2 by ADP—There were differences in the sensitivity to ADP between hGDH1 and hGDH2. Although hGDH1 was activated by ADP at a hyperbolic manner, the ADP stimulatory curves of hGDH2 showed a sigmoidal pattern typically found in allosteric regulation. When the segment 390–448 from the hGDH1 is spliced onto hGDH2 to make hGDH2(hGDH1390–448)hGDH2, it was found to show almost identical sensitivity to ADP regulatory aspects of hGDH1 (Fig. 3B). Similarly, replacing amino acid segment 390–448 of hGDH1 with the corresponding region of hGDH2 to produce chimeric hGDH1(hGDH2390–448)hGDH1 resulted in the activation by ADP at a sigmoidal manner that is comparable with that of wild-type hGDH2 but substantially different from that of the wild-type hGDH1 (Fig. 3B). These results indicate that