Probing the catalytic allosteric mechanism of rabbit muscle pyruvate kinase by tryptophan fluorescence quenching

Li, Feng; Yu, Ting; Zhao, Yuwei; Yu, Shaoning

doi:10.1007/s00249-012-0828-2

Cited by 5 publications

(4 citation statements)

References 30 publications

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“…S99 and K137 of PKM were located in the A and B domains, respectively. Li et al reported that changes in the B domain were more sensitive and important for PKM glycolytic activity because of its high flexibility . In this study, PKM_K137Q showed a lower glycolytic activity compared with the other four PKM variants (Figure A).…”

Section: Discussionmentioning

confidence: 52%

“…Li et al reported that changes in the B domain were more sensitive and important for PKM glycolytic activity because of its high flexibility. 30 In this study, PKM_K137Q showed a lower glycolytic activity compared with the other four PKM variants (Figure 2A). The results of molecular docking suggested that the binding of PKM_K137Q and PEP decreased, which might be a main reason for the low glycolytic activity of PKM_K137Q rather than the binding of PKM_K137Q and ADP.…”

Section: ■ Discussionmentioning

confidence: 52%

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Acetylation and Phosphorylation Regulate the Role of Pyruvate Kinase as a Glycolytic Enzyme or a Protein Kinase in Lamb

Ren,

Li,

et al. 2024

J. Agric. Food Chem.

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Protein post-translational modifications (PTMs) play an essential role in meat quality development. However, the effect of specific PTM sites on meat proteins has not been investigated yet. The characteristics of pyruvate kinase M (PKM) were found to exhibit a close correlation with final meat quality, and thus, serine 99 (S99) and lysine 137 (K137) in PKM were mutated to study their effect on PKM function. The structural and functional properties of five lamb PKM variants, including wild-type PKM (wtPKM), PKM_S99D (S99 phosphorylation), PKM_S99A (PKM S99 dephosphorylation), PKM_K137Q (PKM K137 acetylation), and PKM_K137R (PKM K137 deacetylation), were evaluated. The results showed that the secondary structure, tertiary structure, and polymer formation were affected among different PKM variants. In addition, the glycolytic activity of PKM_K137Q was decreased because of its weakened binding with phosphoenolpyruvate. In the PKM_K137R variant, the actin phosphorylation level exhibited a decrease, suggesting a low kinase activity of PKM_K137R. The results of molecular simulation showed a 42% reduction in the interface area between PKM_K137R and actin, in contrast to wtPKM and actin. These findings are significant for revealing the mechanism of how PTMs regulate PKM function and provide a theoretical foundation for the development of precise meat quality preservation technology.

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Section: Discussionmentioning

confidence: 52%

Section: ■ Discussionmentioning

confidence: 52%

Acetylation and Phosphorylation Regulate the Role of Pyruvate Kinase as a Glycolytic Enzyme or a Protein Kinase in Lamb

Ren,

Li,

et al. 2024

J. Agric. Food Chem.

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“…The domain has been documented to be highly mobile, a characteristic that underpins its expected function within catalysis and regulation. It is proposed that after metal cations coordinate in the active site, the lid domain rotates to allow substrate binding, after which it moves to cover and dehydrate the active site cleft (Li et al 2012;Naithani et al 2015). The position and amount of movement seen in the lid is known to vary depending upon substrate binding within the active site and whether fructose-1,6-phosphate is bound at the regulatory domain (Li et al 2012;Valentini et al 2000).…”

Section: Introductionmentioning

confidence: 99%

“…It is proposed that after metal cations coordinate in the active site, the lid domain rotates to allow substrate binding, after which it moves to cover and dehydrate the active site cleft (Li et al 2012;Naithani et al 2015). The position and amount of movement seen in the lid is known to vary depending upon substrate binding within the active site and whether fructose-1,6-phosphate is bound at the regulatory domain (Li et al 2012;Valentini et al 2000). However, experiments that have provided these insights are generally focused on catalysis (Oria-Hernández et al 2005), regulation (Valentini et al 2000), or deficiencies in human pyruvate kinase mutants (Valentini et al 2002;van Wijk et al 2009), and only involve site directed mutagenesis studies.…”

Section: Introductionmentioning

confidence: 99%

The lid domain is important, but not essential, for catalysis of Escherichia coli pyruvate kinase

et al. 2020

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Pyruvate kinase catalyses the final step of the glycolytic pathway in central energy metabolism.The monomeric structure comprises three domains: a catalytic TIM-barrel, a regulatory domain involved in allosteric activation, and a lid domain that encloses the substrates. The lid domain is thought to close over the TIM-barrel domain forming contacts with the substrates to promote catalysis and may be involved in stabilising the activated state when the allosteric activator is bound. However, it remains unknown whether the lid domain is essential for pyruvate kinase catalytic or regulatory function. To address this, we removed the lid domain of Escherichia coli pyruvate kinase type 1 (PK TIM+Reg ) using protein engineering. Biochemical analyses demonstrate that, despite the absence of key catalytic residues in the lid domain, PK TIM+Reg retains a low-level of catalytic activity and has a reduced binding affinity for the substrate phosphoenolpyruvate. The enzyme retains allosteric activation, but the regulatory profile of the enzyme is changed relative to the wild-type enzyme. Analytical ultracentrifugation and small-angle X-ray scattering data show that, beyond the loss of the lid domain, the PK TIM+Reg structure is not significantly altered and is consistent with the wild type tetramer that is assembled through interactions at the TIM and regulatory domains. Our results highlight the contribution of the lid domain for facilitating pyruvate kinase catalysis and regulation, which could aid in the development of small molecule inhibitors for pyruvate kinase and related lid-regulated enzymes.

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Overestimated accuracy of circular dichroism in determining protein secondary structure

et al. 2013

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Circular dichroism (CD) is a spectroscopic technique widely used for estimating protein secondary structures in aqueous solution, but its accuracy has been doubted in recent work. In the present paper, the contents of nine globular proteins with known secondary structures were determined by CD spectroscopy and Fourier transform infrared spectroscopy (FTIR) in aqueous solution. A large deviation was found between the CD spectra and X-ray data, even when the experimental conditions were optimized. The content determined by FTIR was in good agreement with the X-ray crystallography data. Therefore, CD spectra are not recommended for directly calculating the content of a protein's secondary structure.

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Probing the catalytic allosteric mechanism of rabbit muscle pyruvate kinase by tryptophan fluorescence quenching

Cited by 5 publications

References 30 publications

Acetylation and Phosphorylation Regulate the Role of Pyruvate Kinase as a Glycolytic Enzyme or a Protein Kinase in Lamb

Acetylation and Phosphorylation Regulate the Role of Pyruvate Kinase as a Glycolytic Enzyme or a Protein Kinase in Lamb

The lid domain is important, but not essential, for catalysis of Escherichia coli pyruvate kinase

Overestimated accuracy of circular dichroism in determining protein secondary structure

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