Persistent Binding of MgADP to the E187A Mutant of <i>Escherichia coli</i> Phosphofructokinase in the Absence of Allosteric Effects

Pham, Audrey S.; Janiak‐Spens, Fabiola; Reinhart, Gregory D.

doi:10.1021/bi001768z

Cited by 8 publications

(20 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…While the binding affinity of phosphoglycolate to BsPFK is 10-fold weaker than that of PEP, it has a 3.5-fold greater inhibitory effect on the binding of Fru-6-P (13). Another example of the independence of binding and coupling is the E187A substitution in EcPFK, which leads to the loss of activating effect of MgADP, while the binding of MgADP remains virtually unaffected in this variant (38). Similar behavior was noted for the R252E variant of EcPFK, which can bind both PEP and MgADP, but exhibits neither inhibition by PEP, nor activation by MgADP (39).…”

Section: Discussionmentioning

confidence: 99%

Allosteric Regulation in Phosphofructokinase from the Extreme Thermophile Thermus thermophilus

Lovingshimer

Reinhart

2013

Biochemistry

Self Cite

View full text Add to dashboard Cite

An investigation into the kinetics and regulatory properties of the type-1 phosphofructokinase (PFK) from the extreme thermophile Thermus thermophilus (TtPFK) reveals an enzyme that is inhibited by PEP and activated by ADP by modifying the affinity exhibited for the substrate fructose 6-phosphate (Fru-6-P) in a manner analogous to other prokaryotic PFKs. However, TtPFK binds both of these allosteric ligands significantly more tightly than do other bacterial PFKs while effecting a substantially more modest extent of inhibition or activation at 25°C, reinforcing the principle that binding affinity and effectiveness can be both independent and uncorrelated to one another. These properties have allowed us to rigorously establish that PEP only inhibits by antagonizing the binding of Fru-6-P, and not by influencing turnover – a conclusion that requires kcat be determined under conditions in which both inhibitor and substrate are saturating simultaneously. In addition, the temperature dependence of the allosteric effects on Fru-6-P binding indicate that the coupling free energies are entropy-dominated, as observed previously for PFK from B. stearothermophilus but not for PFK from E. coli, supporting the hypothesis that entropy-dominated allosteric effects may be a characteristic of enzymes derived from thermostable organisms. For such enzymes, the root cause of the allosteric effect may not be easily discerned from static structural information such as might be obtained from X-ray crystallography.

show abstract

Section: Discussionmentioning

confidence: 99%

Allosteric Regulation in Phosphofructokinase from the Extreme Thermophile Thermus thermophilus

Lovingshimer

Reinhart

2013

Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Solution binding assays were performed by single-point fluorescence anisotropy measurements (Koala spectrofluorometer, ISS, Urbana-Champaign, IL). Data were abstracted according to established procedures (34). Briefly, samples were excited with vertically polarized light at 490 nm with a slit width of 8 nm.…”

Section: Fluorescence Anisotropy Measurementsmentioning

confidence: 99%

“…Specifically, equilibrium dissociation constants for the biotin/anti-biotin and DNP/anti-DNP systems were examined in bulk solution and at model membrane surfaces. Solution binding constants were determined by fluorescence anisotropy measurements (32)(33)(34). For surface binding assays, lipid membrane-coated microfluidic devices were employed in conjunction with total internal reflection fluorescence microscopy (TIRFM) (35), a method previously established in our laboratory (36).…”

Section: Introductionmentioning

confidence: 99%

Impact of Hapten Presentation on Antibody Binding at Lipid Membrane Interfaces

Jung

Yang

Lasagna

et al. 2008

Biophysical Journal

Self Cite

View full text Add to dashboard Cite

We report the effects of ligand presentation on the binding of aqueous proteins to solid supported lipid bilayers. Specifically, we show that the equilibrium dissociation constant can be strongly affected by ligand lipophilicity and linker length/structure. The apparent equilibrium dissociation constants (K(D)) were compared for two model systems, biotin/anti-biotin and 2,4-dinitrophenyl (DNP)/anti-DNP, in bulk solution and at model membrane surfaces. The binding constants in solution were obtained from fluorescence anisotropy measurements. The surface binding constants were determined by microfluidic techniques in conjunction with total internal reflection fluorescence microscopy. The results showed that the bulk solution equilibrium dissociation constants for anti-biotin and anti-DNP were almost identical, K(D)(bulk) = 1.7 +/- 0.2 nM vs. 2.9 +/- 0.1 nM. By contrast, the dissociation constant for anti-biotin antibody was three orders of magnitude tighter than for anti-DNP at a lipid membrane interface, K(D) = 3.6 +/- 1.1 nM vs. 2.0 +/- 0.2 microM. We postulate that the pronounced difference in surface binding constants for these two similar antibodies is due to differences in the ligands' relative lipophilicity, i.e., the more hydrophobic DNP molecules had a stronger interaction with the lipid bilayers, rendering them less available to incoming anti-DNP antibodies compared with the biotin/anti-biotin system. However, when membrane-bound biotin ligands were well screened by a poly(ethylene glycol) (PEG) polymer brush, the K(D) value for the anti-biotin antibody could also be weakened by three orders of magnitude, 2.4 +/- 1.1 microM. On the other hand, the dissociation constant for anti-DNP antibodies at a lipid interface could be significantly enhanced when DNP haptens were tethered to the end of very long hydrophilic PEG lipopolymers (K(D) = 21 +/- 10 nM) rather than presented on short lipid-conjugated tethers. These results demonstrate that ligand presentation strongly influences protein interactions with membrane-bound ligands.

show abstract

“…The fact that N59D produced an increase in coupling while making the PEP binding weaker, and A158T and S215H produced a similar increase while having very modest or no effect on the PEP binding, suggests that the binding of the inhibitor and the actual inhibition are independent of one another as we have observed previously. 16 , 17 …”

Section: Resultsmentioning

confidence: 99%

Enhancing Allosteric Inhibition in Thermus thermophilus Phosphofructokinase

Reinhart

2015

Biochemistry

Self Cite

View full text Add to dashboard Cite

The coupling between the binding of the substrate Fru-6-P and the inhibitor phospho(enol)pyruvate (PEP) in phosphofructokinase (PFK) from the extreme thermophile Thermus thermophilus is much weaker than that seen in a PFK from Bacillus stearothermophilus. From the crystal structures of Bacillus stearothermophilus PFK (BsPFK) the residues at positions 59, 158, and 215 in BsPFK are located on the path leading from the allosteric site to the nearest active site and are part of the intricate hydrogen-bonding network connecting the two sites. Substituting the corresponding residues in Thermus thermophilus PFK (TtPFK) with the amino acids found at these positions in BsPFK allowed us to enhance the allosteric inhibition by PEP by nearly 3 kcal mol–1 (50-fold) to a value greater than or equal to the coupling observed in BsPFK. Interestingly, each single variant N59D, A158T, and S215H produced a roughly 1 kcal mol–1 increase in coupling free energy of inhibition. The effects of these variants were essentially additive in the three combinations of double variants N59D/A158T, N59D/S215H, and A158T/S215H as well as in the triple variant N59D/A158T/S215H. Consequently, while the hydrogen-bonding network identified is likely involved in the inhibitory allosteric communication, a model requiring a linked chain of interactions connecting the sites is not supported by these data. Despite the fact that the allosteric activator of the bacterial PFK, MgADP, binds at the same allosteric site, the substitutions at positions 59, 158, and 215 do not have an equally dramatic effect on the binding affinity and the allosteric activation by MgADP. The effect of the S215H and N59D/A158T/S215H substitutions on the activation by MgADP could not be determined because of a dramatic drop in MgADP binding affinity that resulted from the S215H substitution. The single variants N59D and A158T supported binding but showed little change in the free energy of activation by MgADP compared to the wild type TtPFK. These results support previous suggestions that heterotropic inhibition and activation occur by different pathways prokaryotic PFK.

show abstract

Persistent Binding of MgADP to the E187A Mutant of Escherichia coli Phosphofructokinase in the Absence of Allosteric Effects

Cited by 8 publications

References 34 publications

Allosteric Regulation in Phosphofructokinase from the Extreme Thermophile Thermus thermophilus

Allosteric Regulation in Phosphofructokinase from the Extreme Thermophile Thermus thermophilus

Impact of Hapten Presentation on Antibody Binding at Lipid Membrane Interfaces

Enhancing Allosteric Inhibition in Thermus thermophilus Phosphofructokinase

Contact Info

Product

Resources

About