Structure of the Apo Form of <i>Bacillus stearothermophilus</i> Phosphofructokinase

Mosser, R.; Reddy, M. Siva Pratap; Bruning, John B.; Sacchettini, James C.; Reinhart, Gregory D.

doi:10.1021/bi201548p

Cited by 8 publications

(7 citation statements)

References 43 publications

(110 reference statements)

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“…In the present study, we are able to demonstrate unambiguously that the turnover seen for the EA form of the enzyme is the same as that seen for the YEA form, indicating that the binding of PEP does not affect the catalytic activity of TtPFK. Consequently, one can conclude that despite the substantial conformational perturbations introduced by the binding of inhibitor alone − the positioning of residues that define the transition state for the reaction must be unaffected by whether the inhibitor is bound as long as Fru-6-P is present.…”

Section: Discussionmentioning

confidence: 99%

“…The PFKs from Escherichia coli (EcPFK) and Bacillus stearothermophilus (BsPFK) have been extensively studied, resulting in a wealth of kinetic, structural, and thermodynamic information. − The crystal structures of these two enzymes with various ligand combinations bound show a high degree of similarity. However, substantial differences in the binding affinities for the substrate and the allosteric ligands as well as in the magnitude of the allosteric responses are evident.…”

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confidence: 99%

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Allosteric Regulation in Phosphofructokinase from the Extreme Thermophile Thermus thermophilus

Lovingshimer

Reinhart

2013

Biochemistry

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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.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Allosteric Regulation in Phosphofructokinase from the Extreme Thermophile Thermus thermophilus

Lovingshimer

Reinhart

2013

Biochemistry

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“…The analysis of crystal structures of apo, 4 phosphoglycolate-, 5 and Fru-6-P and ADP-bound 6 BsPFK was done using UCSF CHIMERA software.…”

Section: Methodsmentioning

confidence: 99%

“…However, PEP inhibits BsPFK 30-fold more strongly, while MgADP activates both enzymes with nearly equal effectiveness. 1 In an attempt to pinpoint the basis of the weaker coupling between PEP and Fru-6-P exhibited by TtPFK, we have analyzed the available structures, determined by X-ray crystallography, of BsPFK in the apo form, 4 BsPFK bound to the inhibitor phosphoglycolate, 5 and BsPFK bound to both the substrate, fructose 6-phosphate (Fru-6-P) and the allosteric activator (ADP). 6 From these structures one can identify a series of residues involved in an extensive hydrogen-bonding network that extends from the allosteric site to the closest active site.…”

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Enhancing Allosteric Inhibition in Thermus thermophilus Phosphofructokinase

Reinhart

2015

Biochemistry

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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.

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Sodium ions activated phosphofructokinase leading to enhanced d-lactic acid production by Sporolactobacillus inulinus using sodium hydroxide as a neutralizing agent

Zheng

Liu

Sun

et al. 2017

Appl Microbiol Biotechnol

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Sporolactobacillus inulinus is a superior D-lactic acid-producing bacterium and proposed species for industrial production. The major pathway for D-lactic acid biosynthesis, glycolysis, is mainly regulated via the two irreversible steps catalyzed by the allosteric enzymes, phosphofructokinase (PFK) and pyruvate kinase. The activity level of PFK was significantly consistent with the cell growth and D-lactic acid production, indicating its vital role in control and regulation of glycolysis. In this study, the ATP-dependent PFK from S. inulinus was expressed in Escherichia coli and purified to homogeneity. The PFK was allosterically activated by both GDP and ADP and inhibited by phosphoenolpyruvate; the addition of activators could partly relieve the inhibition by phosphoenolpyruvate. Furthermore, monovalent cations could enhance the activity, and Na was the most efficient one. Considering this kind activation, NaOH was investigated as the neutralizer instead of the traditional neutralizer CaCO. In the early growth stage, the significant accelerated glucose consumption was achieved in the NaOH case probably for the enhanced activity of Na-activated PFK. Using NaOH as the neutralizer at pH 6.5, the fermentation time was greatly shortened about 22 h; simultaneously, the glucose consumption rate and the D-lactic acid productivity were increased by 34 and 17%, respectively. This probably contributed to the increased pH and Na-promoted activity of PFK. Thus, fermentations by S. inulinus using the NaOH neutralizer provide a green and highly efficient D-lactic acid production with easy subsequent purification.

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Structure of the Apo Form of Bacillus stearothermophilus Phosphofructokinase

Cited by 8 publications

References 43 publications

Allosteric Regulation in Phosphofructokinase from the Extreme Thermophile Thermus thermophilus

Allosteric Regulation in Phosphofructokinase from the Extreme Thermophile Thermus thermophilus

Enhancing Allosteric Inhibition in Thermus thermophilus Phosphofructokinase

Sodium ions activated phosphofructokinase leading to enhanced d-lactic acid production by Sporolactobacillus inulinus using sodium hydroxide as a neutralizing agent

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