Kinetic properties of pyruvate kinase of rabbit brain

Tsao, Makepeace U.

doi:10.1007/bf00314888

Cited by 10 publications

(5 citation statements)

References 15 publications

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“…No binding of long polar or charged amino acids (Arg, Asn, Asp, Gln, Glu, His, L-homoserine, and Lys) was detected. Consistent with previous studies (11), Pro, Met, Val, and Ile elicit allosteric responses (Figure 4B). Together, these results support the conclusion that the steric bulk, not aromaticity, of the hydrophobic side chain is most important for determining the magnitude of the allosteric inhibition.…”

Section: Resultssupporting

confidence: 93%

“…The single exception to this was Pro. In agreement with earlier studies (11), no binding was apparent for the D-forms of Phe or Ala. The ability to accommodate replacements of the R-hydrogen with other chemical groups was not conclusive.…”

Section: Resultssupporting

confidence: 84%

“…In Figure 3B there is no evidence for the formation of an upper plateau. This is consistent with competitive binding between Ala and Phe and there is no available data that suggests Ala and Phe bind to different binding sites and influence each other's binding allosterically (11). Gly binds competitively with Phe (Table 3).…”

Section: Resultssupporting

confidence: 79%

“…Additions of Ala to Phe-inhibited M 1 -PYK reverse the Phe inhibition (2,10,11). However, small amino acids have not previously been reported to elicit an allosteric response from this enzyme.…”

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confidence: 90%

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Differentiating a Ligand's Chemical Requirements for Allosteric Interactions from Those for Protein Binding. Phenylalanine Inhibition of Pyruvate Kinase^,

et al. 2006

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The isoform of pyruvate kinase from brain and muscle of mammals (M(1)-PYK) is allosterically inhibited by phenylalanine. Initial observations in this model allosteric system indicate that Ala binds competitively with Phe, but elicits a minimal allosteric response. Thus, the allosteric ligand of this system must have requirements for eliciting an allosteric response in addition to the requirements for binding. Phe analogues have been used to dissect what chemical properties of Phe are responsible for eliciting the allosteric response. We first demonstrate that the l-2-aminopropanaldehyde substructure of the amino acid ligand is primarily responsible for binding to M(1)-PYK. Since the allosteric response to Ala is minimal and linear addition of methyl groups beyond the beta-carbon increase the magnitude of the allosteric response, we conclude that moieties beyond the beta-carbon are primarily responsible for allostery. Instead of an all-or-none mechanism of allostery, these findings support the idea that the bulk of the hydrophobic side chain, but not the aromatic nature, is the primary determinant of the magnitude of the observed allosteric inhibition. The use of these results to direct structural studies has resulted in a 1.65 A structure of M(1)-PYK with Ala bound. The coordination of Ala in the allosteric amino acid binding site confirms the binding role of the l-2-aminopropanaldehyde substructure of the ligand. Collectively, this study confirms that a ligand can have chemical regions specific for eliciting the allosteric signal in addition to the chemical regions necessary for binding.

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Section: Resultssupporting

confidence: 93%

Section: Resultssupporting

confidence: 84%

Section: Resultssupporting

confidence: 79%

mentioning

confidence: 90%

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Differentiating a Ligand's Chemical Requirements for Allosteric Interactions from Those for Protein Binding. Phenylalanine Inhibition of Pyruvate Kinase^,

et al. 2006

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“…The possibility that Ala simply destabilizes the protein without binding specifically as an allosteric effector can be argued against by several observations in addition to the cocrystalization of Ala in the allosteric binding site (26). There is a long history of using Ala as a competitive binder when Phe is the allosteric inhibitor (26,(51)(52)(53)(54). Ala binding as a function of PEP concentration has been determined in conjunction with H/DX and SAXS studies (32,42).…”

Section: Temperature As a Variable In Structure And Function Studies ...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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Intermediary Metabolism of Carbohydrates and Other Fuels

Hawkins

Mans

1983

Metabolism in the Nervous System

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Kinetic properties of pyruvate kinase of rabbit brain

Cited by 10 publications

References 15 publications

Differentiating a Ligand's Chemical Requirements for Allosteric Interactions from Those for Protein Binding. Phenylalanine Inhibition of Pyruvate Kinase^,

Differentiating a Ligand's Chemical Requirements for Allosteric Interactions from Those for Protein Binding. Phenylalanine Inhibition of Pyruvate Kinase^,

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

Intermediary Metabolism of Carbohydrates and Other Fuels

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Kinetic properties of pyruvate kinase of rabbit brain

Cited by 10 publications

References 15 publications

Differentiating a Ligand's Chemical Requirements for Allosteric Interactions from Those for Protein Binding. Phenylalanine Inhibition of Pyruvate Kinase,

Differentiating a Ligand's Chemical Requirements for Allosteric Interactions from Those for Protein Binding. Phenylalanine Inhibition of Pyruvate Kinase,

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

Intermediary Metabolism of Carbohydrates and Other Fuels

Contact Info

Product

Resources

About

Differentiating a Ligand's Chemical Requirements for Allosteric Interactions from Those for Protein Binding. Phenylalanine Inhibition of Pyruvate Kinase^,

Differentiating a Ligand's Chemical Requirements for Allosteric Interactions from Those for Protein Binding. Phenylalanine Inhibition of Pyruvate Kinase^,