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PMID: 9023215 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Molecular characterization of glucokinase from Escherichia coli K-12.

Journal of bacteriology ·Vol. 179 ·No. 4 ·1997-02-00 ·Pages 1298-306

Meyer D, Schneider-Fresenius C, Horlacher R, Peist R, Boos W

Abstract

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.

MeSH Terms
Amino Acid Sequence Bacterial Proteins/genetics,metabolism Base Sequence Cloning, Molecular Escherichia coli/enzymology,genetics,growth & development Escherichia coli Proteins Gene Expression Regulation, Bacterial Genes, Bacterial Glucokinase/chemistry,genetics,isolation & purification,metabolism Glucose/metabolism Kinetics Maltose/metabolism Molecular Sequence Data Mutagenesis, Insertional Mutation Recombinant Fusion Proteins/metabolism Repressor Proteins/genetics,metabolism
Chemicals
Bacterial Proteins Escherichia coli Proteins FruR protein, E coli Recombinant Fusion Proteins Repressor Proteins FruR protein, Bacteria Maltose Glucokinase Glucose
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Meyer D
Department of Biology, University of Konstanz, Germany.
Schneider-Fresenius C
Horlacher R
Peist R
Boos W
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.179.4.1298-1306.1997
Published
1997-02-00
Pages
1298-306
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC178829
Subset
IM
External Links
PubMed source DOI: 10.1128/jb.179.4.1298-1306.1997 PMC Full Text View journal details
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