Regulation and Characteristics of Conserved Residues Asp in the Phosphoribosylpyrophosphate Synthetase from Bacillus subtilis
碩士 === 高雄醫學大學 === 生物化學研究所 === 99 === Phosphoribosylpyrophosphate (PRPP) is an important metabolite essential for nucleotide synthesis. PRPP synthetase (EC 2.7.6.1) catalyzes the reaction of ribose 5-phosphate with ATP to yield PRPP and AMP, links the pentose phosphate pathway to the pyrimidine and p...
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ndltd-TW-099KMC051070092015-10-13T20:37:29Z http://ndltd.ncl.edu.tw/handle/46639541447895514205 Regulation and Characteristics of Conserved Residues Asp in the Phosphoribosylpyrophosphate Synthetase from Bacillus subtilis 枯草桿菌中磷酸核糖焦磷酸合成酶的調控與高度保留性之天門冬胺酸特性探討 Ching-Hsin Hsu 許敬鑫 碩士 高雄醫學大學 生物化學研究所 99 Phosphoribosylpyrophosphate (PRPP) is an important metabolite essential for nucleotide synthesis. PRPP synthetase (EC 2.7.6.1) catalyzes the reaction of ribose 5-phosphate with ATP to yield PRPP and AMP, links the pentose phosphate pathway to the pyrimidine and purine nucleotide de novo and salvage pathways, the biosynthesis of histidine and tryptophan, and pyridine nucleotide coenzymes. MgATP acts as the actual substrate of the enzyme. In addition, phosphate and Mg2+ usually act as activators for the activity of bacterial and mammalian PRPP synthetases although SO42- can mimic the effect of phosphate. Both phosphate and SO42- at high concentrations can exhibit an inhibitory effect due to their competitive binding at the R5-P binding site. Sequences alignment suggests that phosphoribosylpyrophosphate binding fingerprint motif is located at residues Asp 223- Thr 231. To investigate the functions of the conserved residues Asp-133, Asp-223, Asp-224, and Asp-227 of Bacillus subtilis PRPP synthetase, we performed site-direct mutagenesis to study the catalysis through the analysis of the steady state kinetics of the reaction and the structural analysis by circular dichroism and the fluorescence spectrometer. Mutants of D133N, D223N, D224N, and D227N were generated, overexpressed in the Escherichia coli strain BL21(DE3) and purified by Ni+ affinity column to homogeneity. The wild-type and mutants of D223N, D224N and D227N show a similar secondary structure based on circular dichroism spectra. To further probe whether the conformation of PRPP synthetase is affected by the mutation on the ribose 5-phosphate binding site, we measured the fluorescence spectrum for the apoenzymes of wild type and mutants of D133N, D223N, D224N, and D227N. The tyrosine fluorescence spectra of wild-type, D223N, and D227N exhibited a single emission λmax at 306nm when excited at 285nm. While tyrosinate moieties fluorescence emission at λmax 337nm was observed for D133N mutant and a double-humped form with both tyrosine and tyrosinate emission spectra was observed for D224N mutant. Steady-state kinetic study shows the activity of PRPP synethetase is regulated by phosphate and sulfate which affects the binding of R 5-P, Mg2+ and MgATP and catalysis. The kinetic parameters for the R5-P in the presence of sulfate and phosphate are 50 and 38 uM in Km, and 25 and 65 s-1 in kcat, respectively. The kinetic parameters Km and kcat of Mg2+ is 1.2mM and 4.9 s-1 in the absence of activator, increase to 0.4 and 0.1mM in Km, and 8 and 12 folds in kcat in the presence of sulfate and phosphate, respectively. The kinetic parameters the S0.5 and kcat of MgATP is 38μM and 10 s-1 in the absence of activator, increase to 55 and 47μM in Km, and 3 and 5 folds in kcat in the presence of sulfate and phosphate, respectively. PRPP synthetase is allosteric enzyme, shows positive cooperativity of MgATP. An addition of the activator sulfate or phosphate affects its cooperativity, resulting from 6 down to 2 nH. Mutant enzymes of D133N, D224N, and D227N increase Km-Mg2+ and decrease kcat. These results indicate that the residues D133, D224 and D227 in the active site play an important role in PRPP synthetase-catalyzed reaction. Finally, we observed recombinant PRPP synthetase has a multiple catalytic promiscuity. Chi-Ching Hwang 黃啟清 2011 學位論文 ; thesis 90 zh-TW |
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碩士 === 高雄醫學大學 === 生物化學研究所 === 99 === Phosphoribosylpyrophosphate (PRPP) is an important metabolite essential for nucleotide synthesis. PRPP synthetase (EC 2.7.6.1) catalyzes the reaction of ribose 5-phosphate with ATP to yield PRPP and AMP, links the pentose phosphate pathway to the pyrimidine and purine nucleotide de novo and salvage pathways, the biosynthesis of histidine and tryptophan, and pyridine nucleotide coenzymes. MgATP acts as the actual substrate of the enzyme. In addition, phosphate and Mg2+ usually act as activators for the activity of bacterial and mammalian PRPP synthetases although SO42- can mimic the effect of phosphate. Both phosphate and SO42- at high concentrations can exhibit an inhibitory effect due to their competitive binding at the R5-P binding site. Sequences alignment suggests that phosphoribosylpyrophosphate binding fingerprint motif is located at residues Asp 223- Thr 231. To investigate the functions of the conserved residues Asp-133, Asp-223, Asp-224, and Asp-227 of Bacillus subtilis PRPP synthetase, we performed site-direct mutagenesis to study the catalysis through the analysis of the steady state kinetics of the reaction and the structural analysis by circular dichroism and the fluorescence spectrometer. Mutants of D133N, D223N, D224N, and D227N were generated, overexpressed in the Escherichia coli strain BL21(DE3) and purified by Ni+ affinity column to homogeneity. The wild-type and mutants of D223N, D224N and D227N show a similar secondary structure based on circular dichroism spectra. To further probe whether the conformation of PRPP synthetase is affected by the mutation on the ribose 5-phosphate binding site, we measured the fluorescence spectrum for the apoenzymes of wild type and mutants of D133N, D223N, D224N, and D227N. The tyrosine fluorescence spectra of wild-type, D223N, and D227N exhibited a single emission λmax at 306nm when excited at 285nm. While tyrosinate moieties fluorescence emission at λmax 337nm was observed for D133N mutant and a double-humped form with both tyrosine and tyrosinate emission spectra was observed for D224N mutant. Steady-state kinetic study shows the activity of PRPP synethetase is regulated by phosphate and sulfate which affects the binding of R 5-P, Mg2+ and MgATP and catalysis. The kinetic parameters for the R5-P in the presence of sulfate and phosphate are 50 and 38 uM in Km, and 25 and 65 s-1 in kcat, respectively. The kinetic parameters Km and kcat of Mg2+ is 1.2mM and 4.9 s-1 in the absence of activator, increase to 0.4 and 0.1mM in Km, and 8 and 12 folds in kcat in the presence of sulfate and phosphate, respectively. The kinetic parameters the S0.5 and kcat of MgATP is 38μM and 10 s-1 in the absence of activator, increase to 55 and 47μM in Km, and 3 and 5 folds in kcat in the presence of sulfate and phosphate, respectively. PRPP synthetase is allosteric enzyme, shows positive cooperativity of MgATP. An addition of the activator sulfate or phosphate affects its cooperativity, resulting from 6 down to 2 nH. Mutant enzymes of D133N, D224N, and D227N increase Km-Mg2+ and decrease kcat. These results indicate that the residues D133, D224 and D227 in the active site play an important role in PRPP synthetase-catalyzed reaction. Finally, we observed recombinant PRPP synthetase has a multiple catalytic promiscuity.
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author2 |
Chi-Ching Hwang |
author_facet |
Chi-Ching Hwang Ching-Hsin Hsu 許敬鑫 |
author |
Ching-Hsin Hsu 許敬鑫 |
spellingShingle |
Ching-Hsin Hsu 許敬鑫 Regulation and Characteristics of Conserved Residues Asp in the Phosphoribosylpyrophosphate Synthetase from Bacillus subtilis |
author_sort |
Ching-Hsin Hsu |
title |
Regulation and Characteristics of Conserved Residues Asp in the Phosphoribosylpyrophosphate Synthetase from Bacillus subtilis |
title_short |
Regulation and Characteristics of Conserved Residues Asp in the Phosphoribosylpyrophosphate Synthetase from Bacillus subtilis |
title_full |
Regulation and Characteristics of Conserved Residues Asp in the Phosphoribosylpyrophosphate Synthetase from Bacillus subtilis |
title_fullStr |
Regulation and Characteristics of Conserved Residues Asp in the Phosphoribosylpyrophosphate Synthetase from Bacillus subtilis |
title_full_unstemmed |
Regulation and Characteristics of Conserved Residues Asp in the Phosphoribosylpyrophosphate Synthetase from Bacillus subtilis |
title_sort |
regulation and characteristics of conserved residues asp in the phosphoribosylpyrophosphate synthetase from bacillus subtilis |
publishDate |
2011 |
url |
http://ndltd.ncl.edu.tw/handle/46639541447895514205 |
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