Kinetic properties of rat liver pyruvate kinase at cellular concentrations of enzyme, substrates and modifiers

Flory, W; Peczon, Benigno D.; Koeppe, Roger E.; Spivey, H. Olin

doi:10.1042/bj1410127

Cited by 37 publications

(14 citation statements)

References 19 publications

(19 reference statements)

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“…Insulin, by reducing cAMP levels, releases the phosphorylation-mediated inhibition and activates PK to promote glycolytic flux (Engstrom, 1978; Feliu et al, 1976). PK is also subjected to allosteric activation by fructose-1,6-BP and inhibition by alanine and ATP (Flory et al, 1974). Fructose-1,6-BP, in addition to allosterically activating PK, also inhibits cAMP-mediated phosphorylation of PK, providing another level of control of PK activity (Pilkis and Granner, 1992).…”

Section: Molecular Mechanisms That Control Hepatic Glucose Productionmentioning

confidence: 99%

Molecular pathophysiology of hepatic glucose production

Sharabi

Tavares

Rines

et al. 2015

Molecular Aspects of Medicine

239

185

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Maintaining blood glucose concentration within a relatively narrow range through periods of fasting or excess nutrient availability is essential to the survival of the organism. This is achieved through an intricate balance between glucose uptake and endogenous glucose production to maintain constant glucose concentrations. The liver plays a major role in maintaining normal whole body glucose levels by regulating the processes of de novo glucose production (gluconeogenesis) and glycogen breakdown (glycogenolysis), thus controlling the levels of hepatic glucose release. Aberrant regulation of hepatic glucose production (HGP) can result in deleterious clinical outcomes, and excessive HGP is a major contributor to the hyperglycemia observed in Type 2 diabetes mellitus (T2DM). Indeed, adjusting glycaemia as close as possible to a non-diabetic range is the foremost objective in the medical treatment of patients with T2DM and is currently achieved in the clinic primarily through suppression of HGP. Here, we review the molecular mechanisms controlling HGP in response to nutritional and hormonal signals and discuss how these signals are altered in T2DM.

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Section: Molecular Mechanisms That Control Hepatic Glucose Productionmentioning

confidence: 99%

Molecular pathophysiology of hepatic glucose production

Sharabi

Tavares

Rines

et al. 2015

Molecular Aspects of Medicine

239

185

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“…M1PYK and M2PYK were purified under identical conditions, and highly purified samples of both isozymes were loaded independently onto a Superdex 200 PC 3.2/30 gel-filtration column. Unless stated otherwise, protein samples were analyzed at physiologically relevant concentrations (0.1 mg/mL) (18). Ten-microliter samples were injected, and the column flow rate was maintained at 0.1 μL min −1 .…”

Section: Methodsmentioning

confidence: 99%

“…2D) and a marked shift in the S 0.5(PEP) value from 0.9 to 0.1 mM, with a concomitant drop in cooperativity (from a n H of 1.2 to 1.0). PEP cellular concentrations are estimated to lie between 0.02 and 0.5 mM (18). Activation of M2PYK by F16BP would therefore increase reaction rates by between four-and 10-fold over this PEP concentration range.…”

Section: M2pyk Exists In Equilibrium Between Inactive Monomer and Activementioning

confidence: 99%

M2 pyruvate kinase provides a mechanism for nutrient sensing and regulation of cell proliferation

Morgan¹,

O’Reilly²,

Wear³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

150

247

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We show that the M2 isoform of pyruvate kinase (M2PYK) exists in equilibrium between monomers and tetramers regulated by allosteric binding of naturally occurring small-molecule metabolites. Phenylalanine stabilizes an inactive T-state tetrameric conformer and inhibits M2PYK with an IC 50 value of 0.24 mM, whereas thyroid hormone (triiodo-L-thyronine, T3) stabilizes an inactive monomeric form of M2PYK with an IC 50 of 78 nM. The allosteric activator fructose-1,6-bisphosphate [F16BP, AC 50 (concentration that gives 50% activation) of 7 μM] shifts the equilibrium to the tetrameric active Rstate, which has a similar activity to that of the constitutively fully active isoform M1PYK. Proliferation assays using HCT-116 cells showed that addition of inhibitors phenylalanine and T3 both increased cell proliferation, whereas addition of the activator F16BP reduced proliferation. F16BP abrogates the inhibitory effect of both phenylalanine and T3, highlighting a dominant role of M2PYK allosteric activation in the regulation of cancer proliferation. X-ray structures show constitutively fully active M1PYK and F16BP-bound M2PYK in an R-state conformation with a lysine at the dimer-interface acting as a peg in a hole, locking the active tetramer conformation. Binding of phenylalanine in an allosteric pocket induces a 13°rotation of the protomers, destroying the peg-in-hole R-state interface. This distinct T-state tetramer is stabilized by flipped out Trp/Arg side chains that stack across the dimer interface. Xray structures and biophysical binding data of M2PYK complexes explain how, at a molecular level, fluctuations in concentrations of amino acids, thyroid hormone, and glucose metabolites switch M2PYK on and off to provide the cell with a nutrient sensing and growth signaling mechanism.allosteric regulation | nutrient sensor | thyroid hormone T3 | Warburg effect T he last of 10 enzymatic steps used to convert glucose to pyruvate is carried out by pyruvate kinase (PYK), which transfers a phosphate from phosphoenolpyruvate to ADP to generate ATP. There are four human PYK isoforms (1); RPYK is restricted to erythrocytes, LPYK is found predominantly in liver and kidney, M1PYK is in muscle and brain, and M2PYK is found in fetal tissues and in proliferating cells. All four isoforms are active as tetramers; M1PYK is constitutively fully active, whereas R-, L-, and M2PYKs are activated by the effector molecule fructose-1,6-bisphosphate (F16BP) (2). M2PYK is a splice variant of the nonallosteric M1PYK isoform and differs by 22 amino acid residues (3). Recent quantification of the concentrations of constitutively fully active M1PYK and allosterically regulated M2PYK isoforms in both cancerous and control tissue samples has revealed that M2PYK is almost always the most abundant isoform in cancer cells, although it can also be predominant in matched control tissues (4). The up-regulation of the M2PYK isoform plays a key role in cancer metabolism (3) and explains the Warburg effect, in which proliferating cancer cells metabolize increas...

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“…Two milliliters of cell suspension containing about 100 mg of cells were shaken (120 strokes/min) in stoppered 20 ml-vials at 370 in the presence of glucose (10 mM, unless otherwise stated), 0.1% bacitracin, and, when added, 10 nM insulin. Bacitracin was added to prevent degradation of the hormones (13 the mixture and allowed to stand for 10 min at 22-24°; the reaction was started by the addition of 0.15 mM P-enolpyruvate.…”

mentioning

confidence: 99%

The Hormonal Control of Pyruvate Kinase Activity and of Gluconeogenesis in Isolated Hepatocytes

Feliu

Hue

Hers

1976

Biochemical Society Transactions

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Treatment of isolated rat hepatocytes with saturating concentrations of glucagon caused several modifications in the kinetic properties of pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40): SO.5 (substrate concentration at half maximum velocity) for phosphoenolpyruvate was about doubled, whereas Vm.., was not changed; the activity measured at 0.15 mM phosphoenolpyruvate (physiological concentration) was reduced 65-80%; and there was also an increase in the Hill coefficient and in the affinity of the enzyme for the inhibitors Mg-ATP and alanine. Glucagon, 3':5'-cyclic AMP, and epinephrine caused an inactivation of pyruvate kinase together with a stimulation of gluconeogenesis. Insulin (10 nM) antagonized the effect of suboptimal doses of glucagon or cyclic AMP and of even maximal doses of epinephrine, on both pyruvate kinase activity and on gluconeogenesis. These observations can be explained by a phosphorylation of pyruvate kinase by cyclic-AMP-dependent protein kinase, as described by Ljungstrom et al. [(1974) Biochim. Biophys. Acta 358, 289-298] in a reconstructed system. They offer a molecular explanation for the hormonal control of gluconeogenesis. Glucose caused an inhibition of gluconeogenesis with no corresponding change in pyruvate kinase activity. The stimulation of gluconeogenesis by glucagon, 3':5'-cyclic AMP, and catecholamines, as well as the antagonistic effect of insulin against small doses of the hyperglycemic agents, has been described by several groups of investigators (for a review, see ref. 1). However, according to recent papers (2, 3), the sites of action of glucagon and catecholamines on hepatic gluconeogenesis are still unknown. A possible site of regulation of gluconeogenesis is at the level of pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) and it has indeed been shown by Krebs and Eggleston (4) that the activity of this enzyme was greatly increased in the livers of rats put on a high carbohydrate diet and was decreased upon fasting, when gluconeogenesis is enhanced. More recently, a rapid inactivation of pyruvate kinase by glucagon in isolated liver preparations has also been briefly reported (5, 6). (12). Two milliliters of cell suspension containing about 100 mg of cells were shaken (120 strokes/min) in stoppered 20 ml-vials at 370 in the presence of glucose (10 mM, unless otherwise stated), 0.1% bacitracin, and, when added, 10 nM insulin. Bacitracin was added to prevent degradation of the hormones (13). The gas phase was 95% 02 and 5% CO2. Glucagon, epinephrine, or cyclic AMP and 2 mM [U-14C]pyruvate, dissolved in isotonic NaCl, were added after 30 min of incubation. Five to 10 min later, 0.1 ml aliquots of the cell suspension were frozen at the temperature of solid CO2 in acetone for the determination of enzyme activities. Twenty minutes after the addition of the labeled precursor, 0.5 ml aliquots of cell suspension were taken in duplicate for the determination of radioactive glucose and glycogen formed from pyruvate.Measurement of Enz...

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Kinetic properties of rat liver pyruvate kinase at cellular concentrations of enzyme, substrates and modifiers

Cited by 37 publications

References 19 publications

Molecular pathophysiology of hepatic glucose production

Molecular pathophysiology of hepatic glucose production

M2 pyruvate kinase provides a mechanism for nutrient sensing and regulation of cell proliferation

The Hormonal Control of Pyruvate Kinase Activity and of Gluconeogenesis in Isolated Hepatocytes

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