Molecular characterization of glucokinase from Escherichia coli K-12

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 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 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.In Escherichia coli and most other bacteria, glucose is transported by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) as glucose-6-phosphate (53), thus eliminating the need for glucokinase in the utilization of glucose. In contrast, the utilization of glucose-containing disaccharides such as lactose, maltose, or trehalose involves the formation of glucose inside the cell and requires its phosphorylation for the effective utilization of the disaccharides. Surprisingly, the presence of a glk mutation (22) does not appear to be a disadvantage in the utilization of these disaccharides. Severe reduction in growth is observed only when, in addition to having the glk mutation, the strain also lacks the ability to phosphorylate glucose via the PTS pathway (14, 59). Two different findings might be relevant for this phenomenon. In the first, glucose and galactose, the products of intracellular ␤-galactosidase action, have been found in large amounts outside the cell after the uptake of lactose, implying that growth on lactose is mediated via the uptake of the secreted products (PTS-mediated phosphorylation in the case of glucose) (35). In the second, internal phosphorylation of glucose by the PTS has been evoked (14). The findings indicate that the enzyme II Glc is also responsible for the utilization of internal glucose.As a consequence, the interest in E. coli glucokinase has been low. Glucokinase activity in E. coli was measured as early as 1953 (21), and MM6, an E. coli mutant defective in the utilization of glucose, was shown to contain normal amounts of glucokinase (4). In that study, the K m of...

Prediction of metabolic fluxes by incorporating genomic context and flux-converging pattern analyses

“…Additionally, glucokinase (GLK) and glucose-6-phosphate isomerase in glycolysis are found in group 5 with reactions in the pentose phosphate (PP) pathway and the Entner-Doudoroff (ED) pathway. In the utilization of glucose, although most of glucose is transported and phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in E. coli and most other bacteria, the expression of glk encoding GLK reaction is still active (36). In contrast to FBA without grouping reaction constraints, which predicts only PTS as a glucose importer, FBA with grouping reaction constraints predicted that both PTS and GLK reactions are in operation, which is consistent with experimental observation.…”

A Quantitative Approach to Catabolite Repression in Escherichia coli

“…Strains and the relevant mutations are listed in Table 1. The glk::cat mutation of BL2 was taken from strain DM1000 (12). The dgsA::Tn10kan mutation was taken from strain KM563, kindly provided by W. Boos (University of Konstanz).…”

Roles of maltodextrin and glycogen phosphorylases in maltose utilization and glycogen metabolism in Corynebacterium glutamicum

“…Like the maltodextrins formed in the course of glycogen degradation, the MalQgenerated maltodextrins are degraded by either MalP or MalZ. Glucose 1-phosphate derived from the GlgP and MalP reactions is converted into glucose 6-phosphate by aphosphoglucomutase (a-Pgm; Lu & Kleckner, 1994;Adhya & Schwartz, 1971), the glucose derived from the MalQ and MalZ reactions is phosphorylated by glucokinase (Glk) to glucose 6-phosphate (Meyer et al, 1997).…”

“…Like the maltodextrins formed in the course of glycogen degradation, the MalQgenerated maltodextrins are degraded by either MalP or MalZ. Glucose 1-phosphate derived from the GlgP and MalP reactions is converted into glucose 6-phosphate by aphosphoglucomutase (a-Pgm; Lu & Kleckner, 1994;Adhya & Schwartz, 1971), the glucose derived from the MalQ and MalZ reactions is phosphorylated by glucokinase (Glk) to glucose 6-phosphate (Meyer et al, 1997).The maltose metabolism of low-GC Gram-positive bacteria, e.g. lactic acid bacteria such as Streptococcus bovis, Lactococcus lactis, Lactobacillus sanfrancensis and Enterococcus faecalis (Ehrmann & Vogel, 1998; Nilsson & Radström, 2001;Le Breton et al, 2005) or the endosporeforming Bacillus subtilis (Schönert et al, 2006), has been thoroughly investigated, and proceeds by different pathways, which, unlike the pathway present in E. coli ( Fig.…”