High affinity binding and allosteric regulation of Escherichia coli glycogen phosphorylase by the histidine phosphocarrier protein, HPr.
The histidine phosphocarrier protein (HPr) is an essential element in sugar transport by the bacterial phosphoenolpyruvate:sugar phosphotransferase system. Ligand fishing, using surface plasmon resonance, was used to show the binding of HPr to a nonphosphotransferase protein in extracts of Escherichia coli; the protein was subsequently identified as glycogen phosphorylase (GP). The high affinity (association constant approximately 10(8) M-1), species-specific interaction was also demonstrated in electrophoretic mobility shift experiments by polyacrylamide gel electrophoresis. Equilibrium ultracentrifugation analysis indicates that HPr allosterically regulates the oligomeric state of glycogen phosphorylase. HPr binding increases GP activity to 250% of the level in control assays. Kinetic analysis of coupled enzyme assays shows that the binding of HPr to GP causes a decrease in the Km for glycogen and an increase in the Vmax for phosphate, indicating a mixed type activation. The stimulatory effect of E. coli HPr on E. coli GP activity is species-specific, and the unphosphorylated form of HPr activates GP more than does the phosphorylated form. Replacement of specific amino acids in HPr results in reduced GP activation; HPr residues Arg-17, Lys-24, Lys-27, Lys-40, Ser-46, Gln-51, and Lys-72 were established to be important. This novel mechanism for the regulation of GP provides the first evidence directly linking E. coli HPr to the regulation of carbohydrate metabolism.
Seok
Y J
YJ
NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Sondej
M
M
Badawi
P
P
Lewis
M S
MS
Briggs
M C
MC
Jaffe
H
H
Peterkofsky
A
A
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D006005:Phosphorylases / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006005:Phosphorylases; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr