Molecular characterization of glucokinase from Escherichia coli K-12.
glk, the structural gene for glucokinase of Escherichia coli, was cloned and sequenced. Overexpression of glk resulted in the synthesis of a cytoplasmic protein with a molecular weight of 35,000. The enzyme was purified, and its kinetic parameters were determined. Its Km values for glucose and ATP were 0.78 and 3.76 mM, respectively. Its Vmax was 158 U/mg of protein. A chromosomal glk-lacZ fusion was constructed and used to monitor glk expression. Under all conditions tested, only growth on glucose reduced the expression of glk by about 50%. A fruR mutation slightly increased the expression of glk-lacZ, whereas the overexpression of plasmid-encoded fruR+ weakly decreased expression. A FruR consensus binding motif was found 123 bp upstream of the potential transcriptional start site of glk. Overexpression of glk interfered with the expression of the maltose system. Repression was strongest in strains that exhibited constitutive mal gene expression due to endogenous induction and, in the absence of a functional MalK protein, the ATP-hydrolyzing subunit of the maltose transport system. It was least effective in wild-type strains growing on maltose or in strains constitutive for the maltose system due to a mutation in malT rendering the mal gene expression independent of inducer. This demonstrates that free internal glucose plays an essential role in the formation of the endogenous inducer of the maltose system.
Meyer
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D
Department of Biology, University of Konstanz, Germany.
Schneider-Fresenius
C
C
Horlacher
R
R
Peist
R
R
Boos
W
W
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005941:Glucokinase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D056968:Lac Repressors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005941:Glucokinase; D005947:Glucose; D008320:Maltose; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C489416:FruR protein, E coli; C068837:FruR protein, Bacteria; D056968:Lac Repressors