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Soo Young Choi - One of the best experts on this subject based on the ideXlab platform.
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Roles of cysteine residues in the inhibition of human glutamate dehydrogenase by palmitoyl-CoA.
Journal of Biochemistry and Molecular Biology, 2012Co-Authors: Seung Cheol Ha, Eun Young Hwang, Soo Young ChoiAbstract:Human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) have been known to be inhibited by palmitoyl-CoA with a high affinity. In this study, we have performed the Cassette Mutagenesis at six different Cys residues (Cys59, Cys93, Cys119, Cys201, Cys274, and Cys323) to identify palmitoyl-CoA binding sites within hGDH2. Four cysteine residues at positions of C59, C93, C201, or C274 may be involved, at least in part, in the inhibition of hGDH2 by palmitoyl-CoA. There was a biphasic relationship, depending on the levels of palmitoyl-CoA, between the binding of palmitoyl-CoA and the loss of enzyme activity during the inactivation process. The inhibition of hGDH2 by palmitoyl-CoA was not affected by the allosteric inhibitor GTP. Multiple Mutagenesis studies on the hGDH2 are in progress to identify the amino acid residues fully responsible for the inhibition by palmitoyl-CoA. [BMB Reports 2012; 45(12): 707-712]
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identification of a udp glucose binding site of human udp glucose dehydrogenase by photoaffinity labeling and Cassette Mutagenesis
Bioconjugate Chemistry, 2005Co-Authors: Myungmin Choi, Seung Ju Yang, Seung Yong Yoon, Soo Young ChoiAbstract:We have identified a UDP-glucose-binding site within human UDP-glucose dehydrogenase (hUGDH) by photoaffinity labeling with a specific probe, [32P]5N3UDP-glucose, and Cassette Mutagenesis using a synthetic hUGDH gene. Photolabel-containing peptides were generated by photolysis followed by tryptic digestion and isolated using the phosphopeptide isolation kit. Photolabeling of these peptides was effectively prevented by the presence of UDP-glucose during photolysis, demonstrating a selectivity of the photoprobe for the UDP-glucose-binding site. Amino acid sequencing and compositional analysis identified the UDP-glucose-binding site of hUGDH as the region containing the sequence, ASVGFGGSXFQK, corresponding to A268−K279 of the amino acid sequence of hUGDH. The unidentified residue, X, can be designated as a photolabeled C276 because the sequences including the cysteine residue in question have a complete identity with those of other UGDH species known. The importance of the C276 residue in the binding of UDP...
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Histidine 454 plays an important role in polymerization of human glutamate dehydrogenase.
FEBS Letters, 2003Co-Authors: Seung Ju Yang, Soo Young Choi, Hyun Jin ChoiAbstract:Abstract Although previous chemical modification studies have suggested several residues to be involved in the maintenance of the quaternary structure of glutamate dehydrogenase (GDH), there are conflicting views for the polymerization process and no clear evidence has been reported yet. In the present study, Cassette Mutagenesis at seven putative positions (Lys333, Lys337, Lys344, Lys346, Ser445, Gly446, and His454) was performed using a synthetic human GDH gene to examine the polymerization process. Of the mutations at the seven different sites, only the Mutagenesis at His454 results in depolymerization of the hexameric GDH into active trimers as determined by HPLC gel filtration analysis and native gradient polyacrylamide gel electrophoresis. The Mutagenesis at His454 has no effects on expression or stability of the protein. The KM values for NADH and 2-oxoglutarate were 1.5-fold and 2.5-fold greater, respectively, for the mutant GDH than for wild-type GDH, indicating that substitution at position 454 had appreciable effects on the affinity of the enzyme for both NADH and 2-oxoglutarate. The Vmax values were similar for wild-type and mutant GDH. The kcat/KM value of the mutant GDH was reduced up to 2.8-fold. The decreased efficiency of the mutant, therefore, results from the increase in KM values for NADH and 2-oxoglutarate. The results with Cassette Mutagenesis and HPLC gel filtration analysis suggest that His454 is involved in the polymerization process of human GDH.
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Importance of glutamate 279 for the coenzyme binding of human glutamate dehydrogenase.
Journal of Biological Chemistry, 2002Co-Authors: Hye-young Yoon, Hyeok Kwon, Soo Young ChoiAbstract:Abstract Although the structure of glutamate dehydrogenase (GDH) has been reported from various sources including mammalian GDH, there are conflicting views regarding the location and mechanism of actions of the coenzyme binding. We have expanded these speculations by photoaffinity labeling and Cassette Mutagenesis. Photoaffinity labeling with a specific probe, [32P]nicotinamide 2-azidoadenosine dinucleotide, was used to identify the NAD+ binding site within human GDH encoded by the synthetic human GDH gene and expressed inEscherichia coli as a soluble protein. Photolabel-containing peptides generated with trypsin were isolated by immobilized boronate affinity chromatography. Photolabeling of these peptides was most effectively prevented by the presence of NAD+ during photolysis, demonstrating a selectivity of the photoprobe for the NAD+ binding site. Amino acid sequencing and compositional analysis identified Glu279 as the site of photoinsertion into human GDH, suggesting that Glu279 is located at or near the NAD+ binding site. The importance of the Glu279 residue in the binding of NAD+ was further examined by Cassette Mutagenesis with mutant enzymes containing Arg, Gly, Leu, Met, or Tyr at position 279. The Mutagenesis at Glu279 has no effects on the expression or stability of the different mutants. The K mvalues for NAD+ were 10–14-fold greater for the mutant GDHs than for wild-type GDH, whereas the V maxvalues were similar for wild-type and mutant GDHs. The efficiency (k cat/K m) of the mutant GDH was reduced up to 18-fold. The decreased efficiency of the mutants results from the increase in K m values for NAD+. In contrast to the K m values for NAD+, wild-type and mutant GDHs show similarK m values for glutamate, indicating that substitution at position 279 had no appreciable effect on the affinity of enzyme for glutamate. There were no differences in sensitivities to ADP activation and GTP inhibition between wild-type and mutant GDH, suggesting that Glu279 is not directly involved in allosteric regulation. The results with photoaffinity labeling and Cassette Mutagenesis studies suggest that Glu279 plays an important role for efficient binding of NAD+ to human GDH.
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identification of the gtp binding site of human glutamate dehydrogenase by Cassette Mutagenesis and photoaffinity labeling
Journal of Biological Chemistry, 2001Co-Authors: Hye-young Yoon, Soo Young ChoiAbstract:Abstract It has been reported that the hyperinsulinism-hyperammonemia syndrome is caused by mutations in glutamate dehydrogenase (GDH) gene that affects enzyme sensitivity to GTP-induced inhibition. To identify the GTP binding site(s) within human GDH, mutant GDHs at Tyr-266 or Lys-450 position were constructed by Cassette Mutagenesis. More than 90% of the initial activities were remained at the concentration of GTP up to 300 μmfor the Lys-450 mutant GDHs regardless of their size, hydrophobicity, and ionization of the side chains, whereas the wild type GDH and the Tyr-266 mutant GDHs were completely inhibited by 30 μmGTP. The binding of GTP to the wild type GDH or the mutant GDHs was further examined by photoaffinity labeling with 8-[γ-32P]azidoguanosine 5′-triphosphate (8-N3-GTP). Saturation of photoinsertion with 8-N3-GTP occurred apparent K d values near 20 μm for the wild type GDH or the Tyr-266 mutant GDH, and the photoinsertion of 8-N3-[γ-32P]GTP was significantly decreased in the presence of 300 μm GTP. Unlike the wild type GDH or the Tyr-266 mutant GDH, less than 10% of photoinsertion was detected in the Lys-450 mutant GDH, and the photoinsertion was not affected by the presence of 300 μm GTP. The results with Cassette Mutagenesis and photoaffinity labeling demonstrate selectivity of the photoprobe for the GTP binding site and suggest that Lys-450, but not Tyr-266, is required for efficient binding of GTP to GDH. Interestingly, studies of the steady-state velocity showed that both the wild type GDH and the Tyr-266 mutant GDHs were inhibited by ATP at concentrations between 10 and 100 μm, whereas less than 10% of the initial activities of the Lys-450 mutant GDHs were diminished by ATP. These results indicate that Lys-450, but not Tyr-266, may be also responsible for the ATP inhibition; therefore, ATP bound to the GTP site.
Seung Ju Yang - One of the best experts on this subject based on the ideXlab platform.
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Regulation of glutamate level in rat brain through activation of glutamate dehydrogenase by Corydalis ternata
Experimental & Molecular Medicine, 2005Co-Authors: Myungmin Choi, Seung Ju Yang, Seung Yong Yoon, Hea-nam HongAbstract:When treated with protopine and alkalized extracts of the tuber of Corydalis ternata for one year, significant decrease in glutamate level and increase in glutamate dehydrogenase (GDH) activity was observed in rat brains. The expression of GDH between the two groups remained unchanged as determined by Western and Northern blot analysis, suggesting a post-translational regulation of GDH activity in alkalized extracts treated rat brains. The stimulatory effects of alkalized extracts and protopine on the GDH activity was further examined in vitro with two types of human GDH isozymes, hGDH1 (house-keeping GDH) and hGDH2 (nerve-specific GDH). Alkalized extracts and protopine activated the human GDH isozymes up to 4.8-fold. hGDH2 (nervespecific GDH) was more sensitively affected by 1 mM ADP than hGDH1 (house-keeping GDH) on the activation by alkalized extracts. Studies with Cassette Mutagenesis at ADP-binding site showed that hGDH2 was more sensitively regulated by ADP than hGDH1 on the activation by Corydalis ternata. Our results suggest that prolonged exposure to Corydalis ternata may be one of the ways to regulate glutamate concentration in brain through the activation of GDH.
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identification of a udp glucose binding site of human udp glucose dehydrogenase by photoaffinity labeling and Cassette Mutagenesis
Bioconjugate Chemistry, 2005Co-Authors: Myungmin Choi, Seung Ju Yang, Seung Yong Yoon, Soo Young ChoiAbstract:We have identified a UDP-glucose-binding site within human UDP-glucose dehydrogenase (hUGDH) by photoaffinity labeling with a specific probe, [32P]5N3UDP-glucose, and Cassette Mutagenesis using a synthetic hUGDH gene. Photolabel-containing peptides were generated by photolysis followed by tryptic digestion and isolated using the phosphopeptide isolation kit. Photolabeling of these peptides was effectively prevented by the presence of UDP-glucose during photolysis, demonstrating a selectivity of the photoprobe for the UDP-glucose-binding site. Amino acid sequencing and compositional analysis identified the UDP-glucose-binding site of hUGDH as the region containing the sequence, ASVGFGGSXFQK, corresponding to A268−K279 of the amino acid sequence of hUGDH. The unidentified residue, X, can be designated as a photolabeled C276 because the sequences including the cysteine residue in question have a complete identity with those of other UGDH species known. The importance of the C276 residue in the binding of UDP...
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Importance of Gly-13 for the coenzyme binding of human UDP-glucose dehydrogenase
Journal of Biological Chemistry, 2004Co-Authors: Jae Wan Huh, Hui Sun Lee, Won Beom Choi, Seung Ju Yang, Hye-young Yoon, Sung Woo ChoAbstract:UDP-glucose dehydrogenase (UGDH) is the unique pathway enzyme furnishing in vertebrates UDP-glucuronate for numerous transferases. In this report, we have identified an NAD(+)-binding site within human UGDH by photoaffinity labeling with a specific probe, [(32)P]nicotinamide 2-azidoadenosine dinucleotide (2N(3) NAD(+)), and Cassette Mutagenesis. For this work, we have chemically synthesized a 1509-base pair gene encoding human UGDH and expressed it in Escherichia coli as a soluble protein. Photolabel-containing peptides were generated by photolysis followed by tryptic digestion and isolated using the phosphopeptide isolation kit. Photolabeling of these peptides was effectively prevented by the presence of NAD(+) during photolysis, demonstrating a selectivity of the photoprobe for the NAD(+)-binding site. Amino acid sequencing and compositional analysis identified the NAD(+)-binding site of UGDH as the region containing the sequence ICCIGAXYVGGPT, corresponding to Ile-7 through Thr-19 of the amino acid sequence of human UGDH. The unidentified residue, X, can be designated as a photolabeled Gly-13 because the sequences including the glycine residue in question have a complete identity with those of other UGDH species known. The importance of Gly-13 residue in the binding of NAD(+) was further examined with a G13E mutant by Cassette Mutagenesis. The Mutagenesis at Gly-13 had no effects on the expression or stability of the mutant. Enzyme activity of the G13E point mutant was not measurable under normal assay conditions, suggesting an important role for the Gly-13 residue. No incorporation of [(32)P]2N(3)NAD(+) was observed for the G13E mutant. These results indicate that Gly-13 plays an important role for efficient binding of NAD(+) to human UGDH.
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Histidine 454 plays an important role in polymerization of human glutamate dehydrogenase.
FEBS Letters, 2003Co-Authors: Seung Ju Yang, Soo Young Choi, Hyun Jin ChoiAbstract:Abstract Although previous chemical modification studies have suggested several residues to be involved in the maintenance of the quaternary structure of glutamate dehydrogenase (GDH), there are conflicting views for the polymerization process and no clear evidence has been reported yet. In the present study, Cassette Mutagenesis at seven putative positions (Lys333, Lys337, Lys344, Lys346, Ser445, Gly446, and His454) was performed using a synthetic human GDH gene to examine the polymerization process. Of the mutations at the seven different sites, only the Mutagenesis at His454 results in depolymerization of the hexameric GDH into active trimers as determined by HPLC gel filtration analysis and native gradient polyacrylamide gel electrophoresis. The Mutagenesis at His454 has no effects on expression or stability of the protein. The KM values for NADH and 2-oxoglutarate were 1.5-fold and 2.5-fold greater, respectively, for the mutant GDH than for wild-type GDH, indicating that substitution at position 454 had appreciable effects on the affinity of the enzyme for both NADH and 2-oxoglutarate. The Vmax values were similar for wild-type and mutant GDH. The kcat/KM value of the mutant GDH was reduced up to 2.8-fold. The decreased efficiency of the mutant, therefore, results from the increase in KM values for NADH and 2-oxoglutarate. The results with Cassette Mutagenesis and HPLC gel filtration analysis suggest that His454 is involved in the polymerization process of human GDH.
Myungmin Choi - One of the best experts on this subject based on the ideXlab platform.
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Regulation of glutamate level in rat brain through activation of glutamate dehydrogenase by Corydalis ternata
Experimental & Molecular Medicine, 2005Co-Authors: Myungmin Choi, Seung Ju Yang, Seung Yong Yoon, Hea-nam HongAbstract:When treated with protopine and alkalized extracts of the tuber of Corydalis ternata for one year, significant decrease in glutamate level and increase in glutamate dehydrogenase (GDH) activity was observed in rat brains. The expression of GDH between the two groups remained unchanged as determined by Western and Northern blot analysis, suggesting a post-translational regulation of GDH activity in alkalized extracts treated rat brains. The stimulatory effects of alkalized extracts and protopine on the GDH activity was further examined in vitro with two types of human GDH isozymes, hGDH1 (house-keeping GDH) and hGDH2 (nerve-specific GDH). Alkalized extracts and protopine activated the human GDH isozymes up to 4.8-fold. hGDH2 (nervespecific GDH) was more sensitively affected by 1 mM ADP than hGDH1 (house-keeping GDH) on the activation by alkalized extracts. Studies with Cassette Mutagenesis at ADP-binding site showed that hGDH2 was more sensitively regulated by ADP than hGDH1 on the activation by Corydalis ternata. Our results suggest that prolonged exposure to Corydalis ternata may be one of the ways to regulate glutamate concentration in brain through the activation of GDH.
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identification of a udp glucose binding site of human udp glucose dehydrogenase by photoaffinity labeling and Cassette Mutagenesis
Bioconjugate Chemistry, 2005Co-Authors: Myungmin Choi, Seung Ju Yang, Seung Yong Yoon, Soo Young ChoiAbstract:We have identified a UDP-glucose-binding site within human UDP-glucose dehydrogenase (hUGDH) by photoaffinity labeling with a specific probe, [32P]5N3UDP-glucose, and Cassette Mutagenesis using a synthetic hUGDH gene. Photolabel-containing peptides were generated by photolysis followed by tryptic digestion and isolated using the phosphopeptide isolation kit. Photolabeling of these peptides was effectively prevented by the presence of UDP-glucose during photolysis, demonstrating a selectivity of the photoprobe for the UDP-glucose-binding site. Amino acid sequencing and compositional analysis identified the UDP-glucose-binding site of hUGDH as the region containing the sequence, ASVGFGGSXFQK, corresponding to A268−K279 of the amino acid sequence of hUGDH. The unidentified residue, X, can be designated as a photolabeled C276 because the sequences including the cysteine residue in question have a complete identity with those of other UGDH species known. The importance of the C276 residue in the binding of UDP...
Mike Merrick - One of the best experts on this subject based on the ideXlab platform.
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Cassette Mutagenesis implicates a helix turn helix motif in promoter recognition by the novel rna polymerase sigma factor σ54
Molecular Microbiology, 1991Co-Authors: J R Coppard, Mike MerrickAbstract:: Cassette Mutagenesis has been used to study the role of a helix-turn-helix (HTH) motif in the novel RNA polymerase sigma factor sigma 54 of Klebsiella pneumoniae. Of the four residues which are predicted to be solvent-exposed in the second helix, the first (Glu-378) tolerated all substitutions, and some mutations of this residue increased expression from sigma 54-dependent promoters. Certain substitutions in the third exposed residue (Ser-382) produced a promoter-specific phenotype and all substitutions in the fourth residue (Arg-383) inactivated the protein, identifying this residue as being likely to be involved in base-specific interactions with the promoter. In vivo footprinting indicated that the inactive HTH mutants of sigma 54 were defective in interaction with both the -24 and -12 regions of the glnAp2 promoter.
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Cassette Mutagenesis implicates a helix‐turn‐helix motif in promoter recognition by the novel RNA polymerase Sigma factor σ54
Molecular Microbiology, 1991Co-Authors: J R Coppard, Mike MerrickAbstract:: Cassette Mutagenesis has been used to study the role of a helix-turn-helix (HTH) motif in the novel RNA polymerase sigma factor sigma 54 of Klebsiella pneumoniae. Of the four residues which are predicted to be solvent-exposed in the second helix, the first (Glu-378) tolerated all substitutions, and some mutations of this residue increased expression from sigma 54-dependent promoters. Certain substitutions in the third exposed residue (Ser-382) produced a promoter-specific phenotype and all substitutions in the fourth residue (Arg-383) inactivated the protein, identifying this residue as being likely to be involved in base-specific interactions with the promoter. In vivo footprinting indicated that the inactive HTH mutants of sigma 54 were defective in interaction with both the -24 and -12 regions of the glnAp2 promoter.
Seung Yong Yoon - One of the best experts on this subject based on the ideXlab platform.
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Regulation of glutamate level in rat brain through activation of glutamate dehydrogenase by Corydalis ternata
Experimental & Molecular Medicine, 2005Co-Authors: Myungmin Choi, Seung Ju Yang, Seung Yong Yoon, Hea-nam HongAbstract:When treated with protopine and alkalized extracts of the tuber of Corydalis ternata for one year, significant decrease in glutamate level and increase in glutamate dehydrogenase (GDH) activity was observed in rat brains. The expression of GDH between the two groups remained unchanged as determined by Western and Northern blot analysis, suggesting a post-translational regulation of GDH activity in alkalized extracts treated rat brains. The stimulatory effects of alkalized extracts and protopine on the GDH activity was further examined in vitro with two types of human GDH isozymes, hGDH1 (house-keeping GDH) and hGDH2 (nerve-specific GDH). Alkalized extracts and protopine activated the human GDH isozymes up to 4.8-fold. hGDH2 (nervespecific GDH) was more sensitively affected by 1 mM ADP than hGDH1 (house-keeping GDH) on the activation by alkalized extracts. Studies with Cassette Mutagenesis at ADP-binding site showed that hGDH2 was more sensitively regulated by ADP than hGDH1 on the activation by Corydalis ternata. Our results suggest that prolonged exposure to Corydalis ternata may be one of the ways to regulate glutamate concentration in brain through the activation of GDH.
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identification of a udp glucose binding site of human udp glucose dehydrogenase by photoaffinity labeling and Cassette Mutagenesis
Bioconjugate Chemistry, 2005Co-Authors: Myungmin Choi, Seung Ju Yang, Seung Yong Yoon, Soo Young ChoiAbstract:We have identified a UDP-glucose-binding site within human UDP-glucose dehydrogenase (hUGDH) by photoaffinity labeling with a specific probe, [32P]5N3UDP-glucose, and Cassette Mutagenesis using a synthetic hUGDH gene. Photolabel-containing peptides were generated by photolysis followed by tryptic digestion and isolated using the phosphopeptide isolation kit. Photolabeling of these peptides was effectively prevented by the presence of UDP-glucose during photolysis, demonstrating a selectivity of the photoprobe for the UDP-glucose-binding site. Amino acid sequencing and compositional analysis identified the UDP-glucose-binding site of hUGDH as the region containing the sequence, ASVGFGGSXFQK, corresponding to A268−K279 of the amino acid sequence of hUGDH. The unidentified residue, X, can be designated as a photolabeled C276 because the sequences including the cysteine residue in question have a complete identity with those of other UGDH species known. The importance of the C276 residue in the binding of UDP...