MOLECULAR CHARACTERIZATION OF GLUCOKINASE FROM ESCHERICHIA-COLI K-12

Glk, the structural gene for glucokinase of Escherichia coli, was clon ed 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 K-m values for glucose and ATP were 0.78 and 3.76 mM, respectively, Its V-max was 158 U/mg of protein, A chromosomal glk-lacZ fusion was constructed an d used to monitor glk expression. Under all conditions tested, only gr owth on glucose reduced the expression of glk by about 50%, A fruR mut ation slightly increased the expression of glk-lacZ, whereas the overe xpression of plasmid-encoded fruR(+) weakly decreased expression. A Fr uR consensus binding motif was found 123 bp upstream of the potential transcriptional start site of glk, Overexpression of glk interfered wi th the expression of the maltose system, Repression was strongest in s trains that exhibited constitutive mal gene expression due to endogeno us induction and, in the absence of a functional MalK protein, the ATP -hydrolyzing subunit of the maltose transport system. It was least eff ective in wild-type strains growing on maltose or in strains constitut ive for the maltose system due to a mutation in MalT rendering the mal gene expression independent of inducer. This demonstrates that free i nternal glucose plays an essential role in the formation of the endoge nous inducer of the maltose system.