Glucose metabolism in Escherichia coli and the effect of increased amount of aldolase.
We present a comparative study of Escherichia coli with normal and increased amounts of fructose-1,6-bisphosphate aldolase. Most experiments employed a resting cell system involving a high cell density (so as to obtain the soluble pool by direct extraction) and anaerobic incubation in the presence of chloramphenicol. Glucose use is linear with time with a rate ca. half of that in growth, fermentation is almost quantitative, and metabolite concentrations reach a quasi steady state. Increased amount of aldolase had little effect on glucose flux; fructose-1,6-P2 concentration decreased by ca. one-third, and the extent of equilibration of its two halves, measured by a dismutation procedure on samples taken during metabolism of [6-14C]glucose, increased from 0.33 [(cpm in C1-3)/(cpm in C1-6)] to 0.43. Using the simplest model, that increased amount of aldolase does not perturb net flux or later metabolites, together with the steady-state rate equations for aldolase and triose-P isomerase, we show that the results with resting cells fit with the extra enzyme being fully active, and do not necessitate special assumptions concerning a glycolytic complex, metabolite compartmentation, or secondary mechanisms assuring high metabolite concentration. However, the fit does require that the measured Vmax values substantially underestimate the actual ones. Calculation also shows that the forms of the predicted curves--and hence the fit with experimental data--of fructose-1,6-P2 concentration and labeling as a function of the amount of aldolase are highly dependent on glyceraldehyde-3-P concentration but independent of the kinetic parameters of aldolase.
Babul
J
J
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
Clifton
D
D
Kretschmer
M
M
Fraenkel
D G
DG
D000693:Anaerobiosis; D002701:Chloramphenicol / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D004099:Dihydroxyacetone Phosphate / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D005285:Fermentation; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D005635:Fructosediphosphates / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D005986:Glyceraldehyde 3-Phosphate / Q000378:metabolism; D007700:Kinetics
D005635:Fructosediphosphates; D005986:Glyceraldehyde 3-Phosphate; D004099:Dihydroxyacetone Phosphate; D002701:Chloramphenicol; D005634:Fructose-Bisphosphate Aldolase; D005947:Glucose; C029063:fructose-1,6-diphosphate