Identification of regions critically affecting kinetics and allosteric regulation of the Escherichia coli ADP-glucose pyrophosphorylase by modeling and pentapeptide-scanning mutagenesis
ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the enzyme responsible for the regulation of bacterial glycogen synthesis. To perform a structure-function relationship study of the Escherichia coli ADP-Glc PPase enzyme, we studied the effects of pentapeptide insertions at different positions in the...
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2007
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00219193_v189_n14_p5325_Ballicora http://hdl.handle.net/20.500.12110/paper_00219193_v189_n14_p5325_Ballicora |
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paper:paper_00219193_v189_n14_p5325_Ballicora2023-06-08T14:43:10Z Identification of regions critically affecting kinetics and allosteric regulation of the Escherichia coli ADP-glucose pyrophosphorylase by modeling and pentapeptide-scanning mutagenesis glucose 1 phosphate adenylyltransferase leucine proline allosterism article binding kinetics catalysis correlation analysis enzyme regulation Escherichia coli gene insertion glycogen synthesis mutagenesis nonhuman pentapeptide scanning mutagenesis priority journal sequence homology stop codon structure activity relation Adenosine Triphosphate Amino Acid Sequence Catalysis Codon, Terminator Escherichia coli Escherichia coli Proteins Genes, Bacterial Glucose-1-Phosphate Adenylyltransferase Kinetics Magnesium Chloride Molecular Sequence Data Mutagenesis, Insertional Oligopeptides Protein Structure, Secondary Protein Structure, Tertiary Sequence Homology, Amino Acid Structural Homology, Protein Structure-Activity Relationship Substrate Specificity Bacteria (microorganisms) Escherichia coli ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the enzyme responsible for the regulation of bacterial glycogen synthesis. To perform a structure-function relationship study of the Escherichia coli ADP-Glc PPase enzyme, we studied the effects of pentapeptide insertions at different positions in the enzyme and analyzed the results with a homology model. We randomly inserted 15 bp in a plasmid with the ADP-Glc PPase gene. We obtained 140 modified plasmids with single insertions of which 21 were in the coding region of the enzyme. Fourteen of them generated insertions of five amino acids, whereas the other seven created a stop codon and produced truncations. Correlation of ADP-Glc PPase activity to these modifications validated the enzyme model. Six of the insertions and one truncation produced enzymes with sufficient activity for the E. coli cells to synthesize glycogen and stain in the presence of iodine vapor. These were in regions away from the substrate site, whereas the mutants that did not stain had alterations in critical areas of the protein. The enzyme with a pentapeptide insertion between Leu102 and Pro103 was catalytically competent but insensitive to activation. We postulate this region as critical for the allosteric regulation of the enzyme, participating in the communication between the catalytic and regulatory domains. Copyright © 2007, American Society for Microbiology. All Rights Reserved. 2007 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00219193_v189_n14_p5325_Ballicora http://hdl.handle.net/20.500.12110/paper_00219193_v189_n14_p5325_Ballicora |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
glucose 1 phosphate adenylyltransferase leucine proline allosterism article binding kinetics catalysis correlation analysis enzyme regulation Escherichia coli gene insertion glycogen synthesis mutagenesis nonhuman pentapeptide scanning mutagenesis priority journal sequence homology stop codon structure activity relation Adenosine Triphosphate Amino Acid Sequence Catalysis Codon, Terminator Escherichia coli Escherichia coli Proteins Genes, Bacterial Glucose-1-Phosphate Adenylyltransferase Kinetics Magnesium Chloride Molecular Sequence Data Mutagenesis, Insertional Oligopeptides Protein Structure, Secondary Protein Structure, Tertiary Sequence Homology, Amino Acid Structural Homology, Protein Structure-Activity Relationship Substrate Specificity Bacteria (microorganisms) Escherichia coli |
| spellingShingle |
glucose 1 phosphate adenylyltransferase leucine proline allosterism article binding kinetics catalysis correlation analysis enzyme regulation Escherichia coli gene insertion glycogen synthesis mutagenesis nonhuman pentapeptide scanning mutagenesis priority journal sequence homology stop codon structure activity relation Adenosine Triphosphate Amino Acid Sequence Catalysis Codon, Terminator Escherichia coli Escherichia coli Proteins Genes, Bacterial Glucose-1-Phosphate Adenylyltransferase Kinetics Magnesium Chloride Molecular Sequence Data Mutagenesis, Insertional Oligopeptides Protein Structure, Secondary Protein Structure, Tertiary Sequence Homology, Amino Acid Structural Homology, Protein Structure-Activity Relationship Substrate Specificity Bacteria (microorganisms) Escherichia coli Identification of regions critically affecting kinetics and allosteric regulation of the Escherichia coli ADP-glucose pyrophosphorylase by modeling and pentapeptide-scanning mutagenesis |
| topic_facet |
glucose 1 phosphate adenylyltransferase leucine proline allosterism article binding kinetics catalysis correlation analysis enzyme regulation Escherichia coli gene insertion glycogen synthesis mutagenesis nonhuman pentapeptide scanning mutagenesis priority journal sequence homology stop codon structure activity relation Adenosine Triphosphate Amino Acid Sequence Catalysis Codon, Terminator Escherichia coli Escherichia coli Proteins Genes, Bacterial Glucose-1-Phosphate Adenylyltransferase Kinetics Magnesium Chloride Molecular Sequence Data Mutagenesis, Insertional Oligopeptides Protein Structure, Secondary Protein Structure, Tertiary Sequence Homology, Amino Acid Structural Homology, Protein Structure-Activity Relationship Substrate Specificity Bacteria (microorganisms) Escherichia coli |
| description |
ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the enzyme responsible for the regulation of bacterial glycogen synthesis. To perform a structure-function relationship study of the Escherichia coli ADP-Glc PPase enzyme, we studied the effects of pentapeptide insertions at different positions in the enzyme and analyzed the results with a homology model. We randomly inserted 15 bp in a plasmid with the ADP-Glc PPase gene. We obtained 140 modified plasmids with single insertions of which 21 were in the coding region of the enzyme. Fourteen of them generated insertions of five amino acids, whereas the other seven created a stop codon and produced truncations. Correlation of ADP-Glc PPase activity to these modifications validated the enzyme model. Six of the insertions and one truncation produced enzymes with sufficient activity for the E. coli cells to synthesize glycogen and stain in the presence of iodine vapor. These were in regions away from the substrate site, whereas the mutants that did not stain had alterations in critical areas of the protein. The enzyme with a pentapeptide insertion between Leu102 and Pro103 was catalytically competent but insensitive to activation. We postulate this region as critical for the allosteric regulation of the enzyme, participating in the communication between the catalytic and regulatory domains. Copyright © 2007, American Society for Microbiology. All Rights Reserved. |
| title |
Identification of regions critically affecting kinetics and allosteric regulation of the Escherichia coli ADP-glucose pyrophosphorylase by modeling and pentapeptide-scanning mutagenesis |
| title_short |
Identification of regions critically affecting kinetics and allosteric regulation of the Escherichia coli ADP-glucose pyrophosphorylase by modeling and pentapeptide-scanning mutagenesis |
| title_full |
Identification of regions critically affecting kinetics and allosteric regulation of the Escherichia coli ADP-glucose pyrophosphorylase by modeling and pentapeptide-scanning mutagenesis |
| title_fullStr |
Identification of regions critically affecting kinetics and allosteric regulation of the Escherichia coli ADP-glucose pyrophosphorylase by modeling and pentapeptide-scanning mutagenesis |
| title_full_unstemmed |
Identification of regions critically affecting kinetics and allosteric regulation of the Escherichia coli ADP-glucose pyrophosphorylase by modeling and pentapeptide-scanning mutagenesis |
| title_sort |
identification of regions critically affecting kinetics and allosteric regulation of the escherichia coli adp-glucose pyrophosphorylase by modeling and pentapeptide-scanning mutagenesis |
| publishDate |
2007 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00219193_v189_n14_p5325_Ballicora http://hdl.handle.net/20.500.12110/paper_00219193_v189_n14_p5325_Ballicora |
| _version_ |
1768544580974673920 |