Phosphofructokinase from Escherichia coli (EcPFK) is allosterically regulated
by MgADP and phospho(enol)pyruvate (PEP). Both molecules compete for binding to
the same allosteric site, however, MgADP activates and PEP inhibits the binding of
fructose-6-phosphate (F6P) to the active site. The mode by which this enzyme can
differentiate between the two ligands and cause the appropriate response is important for
the understanding of the basis of allosteric regulation.
We studied the interactions between an active site and an allosteric site
(heterotropic interactions) within the protein, and found that each of the four unique
heterotropic interactions is unique and the magnitudes of the coupling free energies for
MgADP activation sum up to 100% that of wildtype EcPFK without homotropic
cooperativity in F6P binding. We took on the kinetic and structural characterization of
phosphofructokinase from Lactobacillus bulgaricus (LbPFK) to reveal an enzyme that
exhibits allosteric properties in spite of previous kinetic studies performed by Le Bras et
al. (1991). We have identified residues in EcPFK (Asp59, Gly184 and Asp273), which
are important for the allosteric responses to both MgADP and PEP. Interestingly,
Lys214 is only important in PEP inhibition and not MgADP activation. We can also
differentially disrupt the MgADP heterotropic interactions with the introduction of
G184C within the protein. These results suggest that there are different pathways for
allosteric communication within the enzyme: different paths for MgADP activation and
PEP inhibition, and different paths for each heterotropic interaction with Gly184 being
important for the 33Å MgADP heterotropic interaction.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/1429 |
| Date | 17 February 2005 |
| Creators | Paricharttanakul, Nilubol Monique |
| Contributors | Reinhart, Gregory |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | 6081658 bytes, electronic, application/pdf, born digital |
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