Allosteric Regulation of Pyruvate Kinase M2 Isozyme Involves a Cysteine Residue in the Intersubunit Contact

Ikeda, Yoshitaka; Noguchi, T.

doi:10.1074/jbc.273.20.12227

Cited by 57 publications

(48 citation statements)

References 31 publications

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“…PK activity was calculated at 25 1C by monitoring change of absorbance at 340 nm from 0 to 20 min. The activity of PK here was defined as the quantity of NADH oxidized by 1 mg of lysate protein or recombinant human PKM2 per minute (Ikeda and Noguchi, 1998;Shimada et al, 2008;Vander Heiden et al, 2010). Human recombinant PKM2 was either purchased from BPS Bioscience Inc, with an attached datasheet of characterization (Supplementary Figure 22), or from an in-house source as described in the preceding sections.…”

Section: Pk Activity Assaymentioning

confidence: 99%

Shikonin and its analogs inhibit cancer cell glycolysis by targeting tumor pyruvate kinase-M2

et al. 2011

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We recently reported that shikonin and its analogs were a class of necroptotic inducers that could bypass cancer drug resistance. However, the molecular targets of shikonin are not known. Here, we showed that shikonin and its analogs are inhibitors of tumor-specific pyruvate kinase-M2 (PKM2), among which shikonin and its enantiomeric isomer alkannin were the most potent and showed promising selectivity, that is, shikonin and alkannin at concentrations that resulted in over 50% inhibition of PKM2 activity did not inhibit PKM1 and pyruvate kinase-L (PKL). Shikonin and alkannin significantly inhibited the glycolytic rate, as manifested by cellular lactate production and glucose consumption in drug-sensitive and resistant cancer cell lines (MCF-7, MCF-7/Adr, MCF-7/Bcl-2, MCF-7/Bcl-x L and A549) that primarily express PKM2. HeLa cells transfected with PKM1 showed reduced sensitivity to shikonin-or alkannin-induced cell death. To the best of our knowledge, shikonin and alkannin are the most potent and specific inhibitors to PKM2 reported so far. As PKM2 universally expresses in cancer cells and dictates the last rate-limiting step of glycolysis vital for cancer cell proliferation and survival, enantiomeric shikonin and alkannin may have potential in future clinical application.

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Section: Pk Activity Assaymentioning

confidence: 99%

Shikonin and its analogs inhibit cancer cell glycolysis by targeting tumor pyruvate kinase-M2

et al. 2011

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“…The activities of each of the 11 enzymes were individually measured according to previously reported methods (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23).…”

Section: Measurement Of Enzyme Activity At Saturating Substrate Concementioning

confidence: 99%

Evidence That Does Not Support Pyruvate Kinase M2 (PKM2)-catalyzed Reaction as a Rate-limiting Step in Cancer Cell Glycolysis

Xie

Dai²,

Hu³

2016

Journal of Biological Chemistry

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It has been recognized that the rate-limiting function of pyruvate kinase M2 (PKM2) in glycolysis plays an important role in distributing glycolytic intermediates for anabolic and catabolic purposes in cancer cells. However, after analysis of the catalytic capacity of PKM2 relative to other glycolytic enzymes, the regulation range of PKM2 activity, metabolic flux control, and thermodynamics, we suggest that the PKM2-catalyzed reaction is not a rate-limiting step in cancer cell glycolysis. Hexokinase and phosphofructokinase 1 (PFK1), the first and third enzyme along the pathway, are rate-limiting enzymes that limit the overall glycolytic rate, whereas PKM2 and lactate dehydrogenase, the last two enzymes in the pathway, are for the fast removal of upstream intermediates to prevent the obstruction of the pathway. The argument is in accordance with the catalytic capacity of glycolytic enzymes, regulation range of enzyme activities, metabolic flux control, and thermodynamics.During tumorigenesis, pyruvate kinase (PK) isotype in cells switches from pyruvate kinase M1 (PKM1) or pyruvate kinase L (PKL) to PKM2 (1, 2), suggesting that PKM2 plays a part in tumor initiation and development. PK catalyzes the last step of glycolysis and is commonly regarded as a rate-limiting enzyme. In cancer cells, PKM2 is considered as a rate-limiting enzyme, because its enzyme activity is sensitive to allosteric regulation (3, 4) and it catalyzes a thermodynamically favorable reaction. It is proposed that when it is allosterically inhibited, its catalytic rate is lower than the upstream glycolytic rate; hence, phosphoenolpyruvate (PEP) 4 and upstream glycolytic intermediate may accumulate and flow to anabolic pathways; when it is allosterically activated, its activity is higher than the upstream glycolytic rate, and the upstream glycolytic intermediate would flow to pyruvate, which is then converted to lactate or enters mitochondria for complete oxidation. There are several lines of evidence that support this hypothesis (1, 4 -11).However, the evidence to support PKM2 as a rate-limiting enzyme is not sufficient. If PKM2 is a rate-limiting enzyme, the following lines of evidence are required: (a) PKM2 catalyzes an irreversible reaction; (b) its enzyme activity relative to other glycolytic enzymes is low; (c) its activity is sensitive to regulation; and (d) when its activity is allosterically activated, its catalytic rate should be higher than the upstream glycolytic rate and when its activity is allosterically inhibited, its catalytic rate should be lower than upstream glycolytic rate. So far, there is evidence of points a and c but no b and d.

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“…28 Both M type isoenzymes consist of 531 amino acid residues and differ only in 22 amino acid residues in a exon of 45 amino acids. 4 The isoemzyme specific domains are important for the interaction of the respective pyruvate kinase subunits, 29 and, although structurally very similar, 30-32 the nonallosteric M1 isoenzyme exists only as tetramer while M2-PK is an allosteric enzyme that can exist in two different conformations (a tetrameric and dimeric form). 6,30,[32][33][34] To specifically elucidate the effect of changes in the M2-PK conformation, it was important to design molecules that interact with M2-PK, but not with the closely related M1-PK isoenzyme.…”

Section: M2-pk-binding Peptide Aptamers Discriminate Between Isoenzymesmentioning

confidence: 99%

Isotype‐specific inhibitors of the glycolytic key regulator pyruvate kinase subtype M2 moderately decelerate tumor cell proliferation

Spoden

Mazurek

Morandell

et al. 2008

Intl Journal of Cancer

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Tumor cells express the glycolytic regulator pyruvate kinase subtype M2 (M2-PK), which can occur in a tetrameric form with high affinity to its substrate phosphoenolpyruvate (PEP) and a dimeric form with a low PEP affinity. The transition between both conformations contributes to the control of glycolysis and is important for tumor cell proliferation and survival. Here we targeted M2-PK by synthetic peptide aptamers, which specifically bind to M2-PK and shift the isoenzyme into its low affinity dimeric conformation. The aptamer-induced dimerization and inactivation of M2-PK led to a significant decrease in the PK mass-action ratio as well as ATP:ADP ratio in the target cells. Furthermore, the expression of M2-PK-binding peptide aptamers moderately reduced the growth of immortalized NIH3T3 cell populations by decelerating cell proliferation, but without affecting apoptotic cell death. Moreover, the M2-PK-binding peptide aptamers also reduced the proliferation rate of human U-2 OS osteosarcoma cells. In the present study, we developed the first specific inhibitors of the pyruvate kinase isoenzyme type M2 and present evidence that these inhibitors moderately decelerate tumor cell proliferation. ' 2008 Wiley-Liss, Inc.Key words: tumor metabolism; peptide aptamer; proliferation; pyruvate kinase Tumorigenesis is characterized by an increase in glycolytic enzyme activities as well as distinct changes in the glycolytic isoenzyme equipment. 1-3 A key glycolytic enzyme which is consistently altered in tumor cells is pyruvate kinase (EC 2.7.1.40), catalyzing the dephosphorylation of phosphoenolpyruvate (PEP) to pyruvate, which significantly contributes to net adenosine triphosphate (ATP) production within the glycolytic pathway. Depending on the metabolic functions of various tissues, different isoenzymes of pyruvate kinase are expressed. 4 The pyruvate kinase isoenzyme M1 (M1-PK) is characteristic for tissues with high energy turnover (muscle and brain), whereas the pyruvate kinase isoform L (L-PK) is found in tissues with gluconeogenesis, such as liver. When quiescent cells re-enter the cell cycle, as during tumorigenesis, the tissue-specific isoenzymes disappear and the pyruvate kinase isoenzyme type M2 (M2-PK) is expressed. 3,4 According to the different metabolic functions, M1-PK has the highest affinity to its substrate PEP, whereas the PEP affinity of M2-PK depends on its quaternary structure. M2-PK occurs as a tetrameric form characterized by a high affinity to its substrate PEP which is highly active at physiological PEP concentrations and as a dimeric form with low PEP affinity which is nearly inactive under physiological conditions. 3 The tetrameric but not the dimeric form of M2-PK is associated with other glycolytic enzymes as well as with lactate dehydrogenase (LDH), nucleotide diphosphate kinase and adenylate kinase, within a large complex, referred to as the glycolytic enzyme complex. 5-7 A high amount of the tetrameric form of M2-PK correlates with a relative high PK mass-action ratio ([pyruvate] AE[ATP])...

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Allosteric Regulation of Pyruvate Kinase M2 Isozyme Involves a Cysteine Residue in the Intersubunit Contact

Cited by 57 publications

References 31 publications

Shikonin and its analogs inhibit cancer cell glycolysis by targeting tumor pyruvate kinase-M2

Shikonin and its analogs inhibit cancer cell glycolysis by targeting tumor pyruvate kinase-M2

Evidence That Does Not Support Pyruvate Kinase M2 (PKM2)-catalyzed Reaction as a Rate-limiting Step in Cancer Cell Glycolysis

Isotype‐specific inhibitors of the glycolytic key regulator pyruvate kinase subtype M2 moderately decelerate tumor cell proliferation

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