Determinants of Allosteric Activation of Yeast Pyruvate Kinase and Identification of Novel Effectors Using Computational Screening

Bond, Christopher J.; Jurica, Melissa S.; Mesecar, Andrew D.; Stoddard, Barry

doi:10.1021/bi001443i

Cited by 26 publications

(20 citation statements)

References 27 publications

(42 reference statements)

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“…The presence of a glutamic acid residue at this position was reported as an important property of unregulated PYK isoforms [33]. Moreover, for yeast PYK, it was experimentally shown that a mutation of threonine (T403) to glutamic acid at this structurally conserved position prohibits the allosteric activation of the enzyme by FBP [34].…”

Section: Resultsmentioning

confidence: 99%

Organism-Adapted Specificity of the Allosteric Regulation of Pyruvate Kinase in Lactic Acid Bacteria

et al. 2013

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Pyruvate kinase (PYK) is a critical allosterically regulated enzyme that links glycolysis, the primary energy metabolism, to cellular metabolism. Lactic acid bacteria rely almost exclusively on glycolysis for their energy production under anaerobic conditions, which reinforces the key role of PYK in their metabolism. These organisms are closely related, but have adapted to a huge variety of native environments. They include food-fermenting organisms, important symbionts in the human gut, and antibiotic-resistant pathogens. In contrast to the rather conserved inhibition of PYK by inorganic phosphate, the activation of PYK shows high variability in the type of activating compound between different lactic acid bacteria. System-wide comparative studies of the metabolism of lactic acid bacteria are required to understand the reasons for the diversity of these closely related microorganisms. These require knowledge of the identities of the enzyme modifiers. Here, we predict potential allosteric activators of PYKs from three lactic acid bacteria which are adapted to different native environments. We used protein structure-based molecular modeling and enzyme kinetic modeling to predict and validate potential activators of PYK. Specifically, we compared the electrostatic potential and the binding of phosphate moieties at the allosteric binding sites, and predicted potential allosteric activators by docking. We then made a kinetic model of Lactococcus lactis PYK to relate the activator predictions to the intracellular sugar-phosphate conditions in lactic acid bacteria. This strategy enabled us to predict fructose 1,6-bisphosphate as the sole activator of the Enterococcus faecalis PYK, and to predict that the PYKs from Streptococcus pyogenes and Lactobacillus plantarum show weaker specificity for their allosteric activators, while still having fructose 1,6-bisphosphate play the main activator role in vivo. These differences in the specificity of allosteric activation may reflect adaptation to different environments with different concentrations of activating compounds. The combined computational approach employed can readily be applied to other enzymes.

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Section: Resultsmentioning

confidence: 99%

Organism-Adapted Specificity of the Allosteric Regulation of Pyruvate Kinase in Lactic Acid Bacteria

et al. 2013

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“…K433 binds to the 6′-phosphate oxygen of FBP and is the only residue in the FBP-binding pocket that is different between PKM1 and PKM2 (Dombrauckas et al, 2005; Jurica et al, 1998). Substitution of an analogous FBP-binding residue in yeast PK (T403) by a negatively charged glutamate increased the dissociation constant (K D ) for FBP by 540-fold (Bond et al, 2000). Molecular modeling suggests that acetylation at K433, if it occurs, could significantly neutralize the charge on the lysine side chain, causing a steric hindrance of the FBP binding (Figure 1A).…”

Section: Resultsmentioning

confidence: 99%

Mitogenic and Oncogenic Stimulation of K433 Acetylation Promotes PKM2 Protein Kinase Activity and Nuclear Localization

et al. 2013

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SUMMARY Alternative splicing of the PKM2 gene produces two isoforms, M1 and M2, which are preferentially expressed in adult and embryonic tissues, respectively. The M2 isoform is reexpressed in human cancer and has nonmetabolic functions in the nucleus as a protein kinase. Here, we report that PKM2 is acetylated by p300 acetyltransferase at K433, which is unique to PKM2 and directly contacts its allosteric activator, fructose 1,6-bisphosphate (FBP). Acetylation prevents PKM2 activation by interfering with FBP binding and promotes the nuclear accumulation and protein kinase activity of PKM2. Acetylationmimetic PKM2(K433) mutant promotes cell proliferation and tumorigenesis. K433 acetylation is decreased by serum starvation and cell-cell contact, increased by cell cycle stimulation, epidermal growth factor (EGF), and oncoprotein E7, and enriched in breast cancers. Hence, K433 acetylation links cell proliferation and transformation to the switch of PKM2 from a cytoplasmic metabolite kinase to a nuclear protein kinase.

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“…The primary cause of PEP accumulation has been proposed to be the low activity of Pyk, which uses PEP as a substrate and is present in two isoforms in yeast cells. Pyk1 enzyme activity is stimulated by FBP [37] and Pka-mediated phosphorylation [38]; Pyk2 activity is not activated by FBP, and the gene expression of Pyk2 is subject to glucose repression [39]. Moreover, extremely low activity concomitant with a relatively high transcript level is characteristic of the Pyk2 enzyme [39].…”

Section: Discussionmentioning

confidence: 99%

Effect of the cancer specific shorter form of human 6-phosphofructo-1-kinase on the metabolism of the yeast Saccharomyces cerevisiae

2017

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BackgroundAt first glance, there appears to be a high degree of similarity between the metabolism of yeast (the Crabtree effect) and human cancer cells (the Warburg effect). At the root of both effects is accelerated metabolic flow through glycolysis which leads to overflows of ethanol and lactic acid, respectively. It has been proposed that enhanced glycolytic flow in cancer cells is triggered by the altered kinetic characteristics of the key glycolytic regulatory enzyme 6-phosphofructo-1-kinase (Pfk). Through a posttranslational modification, highly active shorter Pfk-M fragments, which are resistant to feedback inhibition, are formed after the proteolytic cleavage of the C-terminus of the native human Pfk-M. Alternatively, enhanced glycolysis is triggered by optimal growth conditions in the yeast Saccharomyces cerevisiae. ResultsTo assess the deregulation of glycolysis in yeast cells, the sfPFKM gene encoding highly active human shorter Pfk-M fragments was introduced into pfk-null S. cerevisiae. No growth of the transformants with the sfPFKM gene was observed on glucose and fructose. Glucose even induced rapid deactivation of Pfk1 activities in such transformants. However, Pfk1 activities of the sfPFKM transformants were detected in maltose medium, but the growth in maltose was possible only after the addition of 10 mM of ethanol to the medium. Ethanol seemed to alleviate the severely unbalanced NADH/NADPH ratio in the sfPFKM cells. However, the transformants carrying modified Pfk-M enzymes grew faster than the transformants with the human native human Pfk-M enzyme in a narrow ecological niche with a low maltose concentration medium that was further improved by additional modifications. Interestingly, periodic extracellular accumulation of phenylacetaldehyde was detected during the growth of the strain with modified Pfk-M but not with the strain encoding the human native enzyme.ConclusionsHighly active cancer-specific shorter Pfk-M fragments appear to trigger several controlling mechanisms in the primary metabolism of yeast S. cerevisiae cells. These results suggest more complex metabolic regulation is present in S. cerevisiae as free living unicellular eukaryotic organisms in comparison to metazoan human cells. However, increased productivity under broader growth conditions may be achieved if more gene engineering is performed to reduce or omit several controlling mechanisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-017-0362-5) contains supplementary material, which is available to authorized users.

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Determinants of Allosteric Activation of Yeast Pyruvate Kinase and Identification of Novel Effectors Using Computational Screening

Cited by 26 publications

References 27 publications

Organism-Adapted Specificity of the Allosteric Regulation of Pyruvate Kinase in Lactic Acid Bacteria

Organism-Adapted Specificity of the Allosteric Regulation of Pyruvate Kinase in Lactic Acid Bacteria

Mitogenic and Oncogenic Stimulation of K433 Acetylation Promotes PKM2 Protein Kinase Activity and Nuclear Localization

Effect of the cancer specific shorter form of human 6-phosphofructo-1-kinase on the metabolism of the yeast Saccharomyces cerevisiae

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