Abstract
An Escherichia coli B mutant, SG14, accumulates glycogen at 28% the rate observed for the parent E. coli B strain. The glycogen accumulated in the mutant is similar to the glycogen isolated from the parent strain with respect to alpha- and beta-amylosis, chain length determination, and I2-complex absorption spectra. The SG14 mutant contains normal glycogen synthase and branching enzyme activity but has an ADP-glucose pyrophosphorylase with altered kinetic and allosteric properties. The mutant enzyme has been partially purified and requires a 12-fold higher concentration of fructose-P2 or a 26 fold higher concentration of pyridoxal-P than the parent type enzyme for 50% of maximal allosteric activation. TPNH, an effective activator of the E. coli B enzyme, does not activate the SG14 ADP-glucose pyrophosphorylase. Other studies show that for the SG14 enzyme the concentrations of ATP and Mg2+ in the synthesis direction and the concentrations of ADP-glucose and PPi in the pyrophosphorolysis direction required to give 50% of maximal activity are 3- to 6-fold higher than those observed for the parent E. coli B ADP-glucose pyrophosphorylase. The Km for alpha-glucose-1-P at saturating to half-saturating concentrations of the activator, fructose-P2, are about the same for both enzymes. However, in the presence of no activator, the concentration of glucose-1-P required for half-maximal activity is about 1.8-fold higher for the SG14 enzyme. Thus SG14 ADP-glucose pyrophosphorylase has lower affinity for its substrates than does the parent enzyme. Previously the SG14 enzyme had been shown to be less sensitive to inhibition by 5'-AMP than the E. coli B enzyme. This ensensitivity to inhibition renders the SG14 enzyme less responsive to energy charge than the E. coli B ADP-glucose pyrophosphorylase. On the basis of the above results and taking into account the reported concentrations of fructose-P2, of pyridoxal-P, and of the adenine nucleotide pool and its energy charge in E. coli strains, it is concluded that furctose-P2 is the important physiological allosteric activator of E. coli ADP-glucose pyrophosphorylase. Furthermore, the 1.7-fold increased rate of accumulation of glycogen observed when E. coli B or SG14 shifts from exponential phase to stationary phase of growth in nitrogen-limiting media can be accounted for by the 2.4-fold increase of the levels of the glycogen biosynthetic enzymes, glycogen synthase, and ADP-glucose pyrophosphorylase. Thus both allosteric regulation of the ADP-glucose pyrophosphorylase as well as the genetic regulation of the biosynthesis of the glycogen biosynthetic enzymes are involved in the regulation of glycogen accumulation in E. coli B.
Highlights
Other studies show that for the SG14 enzyme the concentrations of ATP and Mg2+ in the synthesis direction and the concentrations of ADP-glucose and PP, in the pyrophosphorolysis direction required to give 50% of maximal activity are 3- to g-fold higher than those observed for the parent E. coli B ADP-glucose pyrophosphorylase
Optimal activity for pyrophosphorolysis in Hepes buffer is observed between pH 7.5 to 8.0 for SG14 enzyme and 6.5 to 7.5 for E. coli B enzyme in activated and unactivated conditions
Whereas the maximal rate of ADP-glucose synthesis is equal to the maximal rate of pyrophosphorolysis with the Escherichia coli B enzyme at pH 7.0 (Hepes) and pH 8.5 (Tris), the rates of synthesis of ADP-glucose catalyzed by the SGl4 ADP-glucose pyrophosphorylase is only 25% to 27% the rate of ADP-glucose pyrophosphorolysis observed either at pH 7.0 (Hepes) or pH 8.5 (Tris)
Summary
For maximal activity of the E. coli B enzyme the reaction mixture contained 0.3 rmol of ATP, 1.0 Fmol of MgCl,, and 0.3 ymol of fructose-P,. Maximal velocity of the E. coli B enzyme was measured with 0.2 pmol of ADP-glucose, 2 rmol of MgCl, and 0.3 rmol of fructose-P* in the reaction mixture. The absence of activator, optimal rates of pyrophosphorolysis for the SG14 enzyme were achieved with 1.5 pmol of ADP-glucose and 3.0 Fmol of MgCl, in the above reaction mixture. For measuring maximal activity for the E. coli B enzyme, the amounts of ADP-glucose and MgCl, were 0.7 and 5 Fmol, respectively. Titers of ADP-glucose pyrophosphorylase (pyrophosphorylase direction under maximal activated conditions with fructose-l’, as activator) and bacterial glycogen synthase were measured in uncentrifuged crude extracts as previously described [4]. Measurement of iodine comulex spectra was done as descrided [31]
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