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 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 Leu(102) and Pro(103) 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.
Ballicora
Miguel A
MA
Department of Chemistry, Loyola University, Chicago, IL 60626, USA. mballic@luc.edu
Erben
Esteban D
ED
Yazaki
Terutaka
T
Bertolo
Ana L
AL
Demonte
Ana M
AM
Schmidt
Jennifer R
JR
Aleanzi
Mabel
M
Bejar
Clarisa M
CM
Figueroa
Carlos M
CM
Fusari
Corina M
CM
Iglesias
Alberto A
AA
Preiss
Jack
J
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D002384:Catalysis / Q000187:drug effects; D018388:Codon, Terminator / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D051599:Glucose-1-Phosphate Adenylyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009842:Oligopeptides / Q000235:genetics*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D018388:Codon, Terminator; D029968:Escherichia coli Proteins; D009842:Oligopeptides; D015636:Magnesium Chloride; D000255:Adenosine Triphosphate; D051599:Glucose-1-Phosphate Adenylyltransferase