Changes in the allosteric site of human liver pyruvate kinase upon activator binding include the breakage of an intersubunit cation–π bond

McFarlane, Jeffrey S.; Ronnebaum, Trey; Meneely, Kathleen M.; Chilton, Annemarie S.; Fenton, Aron W.; Lamb, Audrey L.

doi:10.1107/s2053230x19007209

Cited by 13 publications

(20 citation statements)

References 21 publications

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“…By having a method that reports on many functions at once, we have found hLPYK to be a useful model to evaluate individual amino acid positions based on functional outcomes due to substitution 2,3 . In particular, our past studies have identified substitutions at nonconserved positions in and near the allosteric binding sites that modulated multiple functional parameters 2,3,5‐9 (Figure 1, green dots).…”

Section: Resultsmentioning

confidence: 99%

“…We have been using human liver pyruvate kinase (hLPYK) to probe the functional roles of nonconserved protein positions. In our studies, substitutions at nonconserved positions in and near the allosteric binding sites modulated multiple functional parameters 2,3,5‐9 . To better understand how the nonconserved positions contribute to function, we wish to compare their biochemical, biophysical and structural characteristics to those of nonconserved positions that have little effect on function.…”

Section: Introductionmentioning

confidence: 99%

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Identification of biochemically neutral positions in liver pyruvate kinase

Martin

Tang

et al. 2020

Proteins

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Understanding how each residue position contributes to protein function has been a long‐standing goal in protein science. Substitution studies have historically focused on conserved protein positions. However, substitutions of nonconserved positions can also modify function. Indeed, we recently identified nonconserved positions that have large substitution effects in human liver pyruvate kinase (hLPYK), including altered allosteric coupling. To facilitate a comparison of which characteristics determine when a nonconserved position does vs does not contribute to function, the goal of the current work was to identify neutral positions in hLPYK. However, existing hLPYK data showed that three features commonly associated with neutral positions—high sequence entropy, high surface exposure, and alanine scanning—lacked the sensitivity needed to guide experimental studies. We used multiple evolutionary patterns identified in a sequence alignment of the PYK family to identify which positions were least patterned, reasoning that these were most likely to be neutral. Nine positions were tested with a total of 117 amino acid substitutions. Although exploring all potential functions is not feasible for any protein, five parameters associated with substrate/effector affinities and allosteric coupling were measured for hLPYK variants. For each position, the aggregate functional outcomes of all variants were used to quantify a “neutrality” score. Three positions showed perfect neutral scores for all five parameters. Furthermore, the nine positions showed larger neutral scores than 17 positions located near allosteric binding sites. Thus, our strategy successfully enriched the dataset for positions with neutral and modest substitutions.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Identification of biochemically neutral positions in liver pyruvate kinase

Martin

Tang

et al. 2020

Proteins

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“…The L and R isozymes are dominant in the liver and erythrocytes respectively, and both forms are allosterically regulated by FBP. Recently, in human liver PyK switching of the allosteric loop from an open to closed conformation in the presence of FBP has been attributed to the change in interactions at the C–C interface which leads to the altered substrate affinity of the enzyme . Muscle specific isoforms, PyK‐M1 and PyK‐M2, are expressed from the same gene but differ in 22 residues located within a 56 residue region situated at the C–C interface close to the allosteric effector‐binding site.…”

Section: Structurally and Functionally Distinct Pyksmentioning

confidence: 99%

“…Recently, in human liver PyK switching of the allosteric loop from an open to closed conformation in the presence of FBP has been attributed to the change in interactions at the C-C interface which leads to the altered substrate affinity of the enzyme. 76 Muscle specific isoforms, PyK-M1 and PyK-M2, are expressed from the same gene but differ in 22 residues located within a 56 residue region situated at the C-C interface close to the allosteric effectorbinding site. PyK-M1 is a highly active nonallosteric form found in tissues, such as heart, brain, and skeletal muscle that need to rapidly generate large amounts of ATP.…”

Section: Vertebrate Pyk Isozymesmentioning

confidence: 99%

An overview of structure, function, and regulation of pyruvate kinases

et al. 2019

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In the last step of glycolysis Pyruvate kinase catalyzes the irreversible conversion of ADP and phosphoenolpyruvate to ATP and pyruvic acid, both crucial for cellular metabolism. Thus pyruvate kinase plays a key role in controlling the metabolic flux and ATP production. The hallmark of the activity of different pyruvate kinases is their tight modulation by a variety of mechanisms including the use of a large number of physiological allosteric effectors in addition to their homotropic regulation by phosphoenolpyruvate. Binding of effectors signals precise and orchestrated movements in selected areas of the protein structure that alter the catalytic action of these evolutionarily conserved enzymes with remarkably conserved architecture and sequences. While the diverse nature of the allosteric effectors has been discussed in the literature, the structural basis of their regulatory effects is still not well understood because of the lack of data representing conformations in various activation states. Results of recent studies on pyruvate kinases of different families suggest that members of evolutionarily related families follow somewhat conserved allosteric strategies but evolutionarily distant members adopt different strategies. Here we review the structure and allosteric properties of pyruvate kinases of different families for which structural data are available.

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“…To support this idea, first consider that many pyruvate kinase isozymes are regulated by phosphorylated sugars (55), although the identity of those effectors varies considerably among isozymes (56). Our previous study used extensive numbers of effector analogs, point mutations, and crystallographically determined structures to characterize which regions of the fructose-1,6-bisphosphate (Fru-1,6-BP) allosteric site of human liver PYK (LPYK) contribute to allostery (50,57). One of the two PYK isozymes from Escherichia coli (EcPYK) is also regulated by Fru-1,6-BP (58).…”

Section: The Influence Of Silent Coupling On the Evolution Of An Allo...mentioning

confidence: 99%

H/D Exchange Characterization of Silent Coupling: Entropy-Enthalpy Compensation in Allostery

Prasannan

Gmyrek

Martin

et al. 2020

Biophysical Journal

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The allosteric coupling constant in K-type allosteric systems is defined as a ratio of the binding of substrate in the absence of effector to the binding of the substrate in the presence of a saturating concentration of effector. As a result, the coupling constant is itself an equilibrium value comprised of a DH and a TDS component. In the scenario in which TDS completely compensates DH, no allosteric influence of effector binding on substrate affinity is observed. However, in this ''silent coupling'' scenario, the presence of effector causes a change in the DH associated with substrate binding. A suggestion has now been made that ''silent modulators'' are ideal drug leads because they can be modified to act as either allosteric activators or inhibitors. Any attempt to rationally design the effector to be an allosteric activator or inhibitor is likely to be benefitted by knowledge of the mechanism that gives rise to coupling. Hydrogen/deuterium exchange with mass spectrometry detection has now been used to identify regions of proteins that experience conformational and/or dynamic changes in the allosteric regulation. Here, we demonstrate the expected temperature dependence of the allosteric regulation of rabbit muscle pyruvate kinase by Ala to demonstrate that this effector reduces substrate (phosphoenolpyruvate) affinity at 35 C and at 10 C but is silent at intermediate temperatures. We then explore the use of hydrogen/deuterium exchange with mass spectrometry to evaluate the areas of the protein that are modified in the mechanism that gives rise to the silent coupling between Ala and phosphoenolpyruvate. Many of the peptide regions of the protein identified as changing in this silent system (Ala as the effector) were included in changes previously identified for allosteric inhibition by Phe.

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Changes in the allosteric site of human liver pyruvate kinase upon activator binding include the breakage of an intersubunit cation–π bond

Cited by 13 publications

References 21 publications

Identification of biochemically neutral positions in liver pyruvate kinase

Identification of biochemically neutral positions in liver pyruvate kinase

An overview of structure, function, and regulation of pyruvate kinases

H/D Exchange Characterization of Silent Coupling: Entropy-Enthalpy Compensation in Allostery

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