Abstract
Treatment of nitrogen-starved cultures of Escherichia coli W4597(K) with sodium azide results in simultaneous changes in both glucose 6-phosphate and fructose 1,6-diphosphate as well as in the rate of glycogen synthesis. Based on these observations, a comprehensive equation was developed which relates the cellular levels of both of these hexose phosphates with the rate of glycogen synthesis. This relationship apparently represents the interaction in vivo between the rate-limiting enzyme of bacterial glycogen synthesis, glucose 1-phosphate adenylyltransferase (adenosine diphosphoglucose synthetase, EC 2.7.7.27), and its substrate glucose 1-phosphate (reflected by glucose 6-phosphate) and its major allosteric activator fructose diphosphate. The form of the equation that describes this relationship was determined from studies presented here of the kinetic properties of the E. coli W4597(K) enzyme in the presence of physiological concentrations of its substrates and modulators. We show here and in subsequent reports of this series that the comprehensive relationship between glycogen synthesis and hexose phosphates can serve as a reference to evaluate the possible participation of new factors in the regulation of glycogen synthesis. Treatment with NaN3 did not change the cellular level of glucose 1-phosphate adenylyltransferase. The value of the adenylate energy charge, (ATP + 1/2 ADP)/(ATP + ADP + AMP), was maintained despite losses of up to 35% in cellular adenylates. The quantitative co-variance between hexose phosphates and the cellular rate of glucose utilization that we previously described for other metabolic conditions was also observed in the azide-treated cultures. We integrate the new information into the system of coordinated regulation of glycogen synthesis, glycolysis, and glucose utilization that we proposed previously.
Highlights
We have shown that changes in the rate of glycogen synthesis in the intact Escherichia coli cell parallel changes in the cellular level of the energy charge, fructose-Pz or glucose
Is reflected in uiuo by the concentration of glucose-6-P.) In order to study the regulation of glycogen synthesis in a broader range of conditions than in our previous studies, we found it necessary to develop a single quantitative relationship which correlated the cellular rate of glycogen synthesis and the cellular levels of fructose-PL, and glucose-l-P at a given value of the energy charge
Regulation of Glycogenesis and Glucose Use in E. coli results were presented previously [14, 15].) We integrate the new information obtained from the effects of NaNZ1 on cellular metabolism into the system of coordinated regulation of glycogen synthesis, glycolysis, and glucose utilization that we proposed previously [12]
Summary
Kinetic Properties of E. coli W4597(K) ADP-Glucose Synthetase at Energy Charge 0.87. Preliminary studies of enzyme determinations at energy charge 0.87 showed that the concentrations of adenine nucleotides were not altered during a lo-min incubation with up to 10 pg of cellular protein. (In a lo-min incubation with 20 or 30 kg the value of the energy charge decreased to 0.85 or 0.84, respectively.) enzyme activities at energy charge 0.87 were determined using no more than 10 pg of cellular protein/. Glucose-l-P and fructose-Pz, over the ranges of concentrations studied here, do not appear to alter the interaction of the other hexose phosphate with the W4597(K) enzyme. The value of V in Equation 3 was calculated from the measured velocities obtained with various concentrations of glucose-1-P and fructose-P2 (Table II) using the values of the SOA, ACM, and n for the W4597(K) enzyme determined in the preceding experiments. The velocity measured at energy charge 1.0 (that is, in the absence of the inhibitor, AMP) in the presence of concentrations of glucose-l-P and fructose-P,, that are essentially saturating at energy charge 1.0 is 1910 pmol/g of protein/h (Table II). The values of the maximal velocity (V) were calculated from the equation using the enzyme activities (v) and the concentrations of glucose-l-P and fructose-P:!
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