JBC, Vol. 250, Issue 19, 7631-7638, Oct, 1975
Biosynthesis of bacterial glycogen. Kinetic studies of a glucose-1-phosphate adenylyltransferase (EC 2.7.7.27) from a glycogen-deficient mutant of Escherichia coli B
J. Preiss, E. Greenberg and A. Sabraw
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.
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