Comparative kinetic studies on the L-type pyruvate kinase from rat liver and the enzyme phosphorylated by cyclic 3′,5′-AMP-stimulated protein kinase

Ekman, Pia; Dahlqvist, Ulla; Humble, Elisabet; Engström, Lorentz

doi:10.1016/0005-2744(76)90285-0

Cited by 94 publications

(29 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The decrease in the PEP transformation rate in the phosphorylation switch of L-PK has so far been reported to be only 9 -6 -fold by Ekman et al [27], 5.6 -4.8 -fold by El-Maghrabi et al [11] or 11.6 -8.5 -fold by Schworer et al [14]. The present results demonstrate that the real scale of the switch should exceed 500 -fold.…”

Section: Some Physiological Implicationssupporting

confidence: 43%

Phosphorylation is switch of L-type pyruvate kinase allostery

et al. 2010

View full text Add to dashboard Cite

Among four pyruvate kinase isoenzymes, M1, M2, R and L, only M1 is considered as a nonallosteric enzyme. However, here we show that the non-phosphorylated L-type pyruvate kinase (L-PK) is also a non-allosteric enzyme with respect to its substrate phosphoenolpyruvate (PEP). The allosteric catalytic properties of L-PK are switched on through phosphorylation by cAMP-dependent protein kinase. The non-phosphorylated enzyme was produced by expressing the rat L-PK in E. coli, as the bacterium does not have mammalian-type protein kinases. The resulting tetrameric protein was phosphorylated with a stoichiometric ratio of one mole of phosphate per one L-PK monomer. Activity of the phosphorylated enzyme was allosterically regulated by PEP with the Hill coefficient n=2.5. It was observed that allostery was engaged by phosphorylation of the first subunit in the tetrameric enzyme, while further phosphorylation only modulated this effect. The discovered switching between non-allosteric and allosteric forms of L-PK and the possibility of modulating the allostery by phosphorylation are important for understanding of the interrelationship between allostery and the regulatory phosphorylation in general, and may have implication for further analysis of glycolysis regulation in the liver.© Versita Sp. z o.o. Keywords: Regulatory phosphorylation • Allostery • L-type pyruvate kinase • Switch between allosteric and non-allosteric forms • Glycolysis regulation

show abstract

Section: Some Physiological Implicationssupporting

confidence: 43%

Phosphorylation is switch of L-type pyruvate kinase allostery

et al. 2010

View full text Add to dashboard Cite

show abstract

“…This indicates that a change in kinetic properties of pyruvate kinase also occurs in the intact animal stressing its physiological importance. The change in kinetic parameters introduced by glucagon incubation of hepatocytes and starvation of the intact animal are similar and idenkal to the changes observed on in vitro phosphorylation of the enzyme purified from rat liver [Ekman et al (1976) 3. The glucagon inhibited enzyme is partially reactivated irz vitro by incubation at 37 "C. This reactivation is promoted by the presence of 10 mM MgZf.…”

mentioning

confidence: 55%

Hormone‐Induced Changes in Pyruvate Kinase

Berkel

Kruijt

Koster

1977

European Journal of Biochemistry

View full text Add to dashboard Cite

1. The kinetic properties of pyruvate kinase from isolated hepatocytes of fed rat liver incubated with and without glucagon (1 pM) are compared. Glucagon incubation of hepatocytes decreases the apparent affinities of the enzyme for the substrate phosphoeizolpyruvate and the allosteric activator fructose 1,6-bisphosphate while the apparent Ki values for alanine and ATP are lowered.The K, for the second substrate ADP is not influenced by glucagon incubation. The kinetic curves show intermediate plateaus indicative of the presence of two enzyme forms, an active form (called La) and a less active form (called Lb). It is concluded that the effect of glucagon on the kinetic parameters of pyruvate kinase is caused by a shift of ihe equilibrium between both enzyme forms to the less active Lb side.2. Comparison of the kinetic parameters of pyruvate kinase of a total homogenate from a fed and a 48-h-starved rat indicates that starvation causes a decrease of the apparent affinities of the enzyme for phosphoenolpyruvate and fructose 1,6-bisphosphate while the apparent K i values for alanine and ATP are lowered. The Km for ADP is not influenced by starvation. This indicates that a change in kinetic properties of pyruvate kinase also occurs in the intact animal stressing its physiological importance. The change in kinetic parameters introduced by glucagon incubation of hepatocytes and starvation of the intact animal are similar and idenkal to the changes observed on in vitro phosphorylation of the enzyme purified from rat liver [Ekman et al. (1976) 3. The glucagon inhibited enzyme is partially reactivated irz vitro by incubation at 37 "C. This reactivation is promoted by the presence of 10 mM MgZf. The reactivation in vitro can be blocked completely by 2 0 m M KF. These properties of reactivation are most consistent with a protein-phosphatase-mediated process. The reactivation is inhibited by a high Ca2 + concentration (2.6 mM).4. Incubation in &ro of a liver homogenate of a fed and starved rat with 10 mM Mg2+ + 0.5 mM ATP in the presence of F -shows that starvation increases the pyruvate kinase inactivation reaction presumably due to protein kinase activity.5. It is concluded that hormonal regulation of pyruvate kinase activity is exerted both by changing the degree of (de)phosphorylation of the enzyme and by changing the concentration of the allosteric activator fructose 1,6-bisphosphate which dcpends, at least in part, on the hormonal control of the phosphofructokinase -fructose-l,6-bisphosphatase cycle.Pyruvate kinase catalyzes the last step of glycolysis.

show abstract

“…Indeed, the carbohydrate-rich diet stimulates the transcription of the L-type pyruvate kinase gene and stabilizes its specific mRNAs (35, 4, 5), whereas glucose feeding and insulin block the transcription of the PEP carboxykinase gene and shorten the half-life of its mRNAs (13,36,37). Conversely, glucagon via its second messenger, cAMP, blocks L-type pyruvate kinase gene transcription, shortens the t1/2 of the mRNA, and inactivates the enzyme by reversible phosphorylation at its catalytic site (4)(5)(6)(7)(8)(9). Glucagon also stimulates PEP carboxykinase gene transcription and enhances the specific mRNA stability (12,36).…”

Section: Resultsmentioning

confidence: 99%

“…Glucagon, for instance, rapidly blocks transcription of the L-type pyruvate kinase gene, shortens the t1/2 of its mRNA, and inactivates the mature enzyme by phosphorylation via its second messenger, cAMP (4)(5)(6)(7)(8)(9). Glucagon simultaneously switches on the transcription of the PEP carboxykinase gene so as to produce an increase in glucose production by the liver during fasting (10)(11)(12).…”

Section: Introductionmentioning

confidence: 99%

In vivo regulation of glycolytic and gluconeogenic enzyme gene expression in newborn rat liver.

Lyonnet¹,

Coupé²,

Girard³

et al. 1988

J. Clin. Invest.

View full text Add to dashboard Cite

Glucagon and its second messenger, cAMP, are known to rapidly block expression of the L-type pyruvate kinase gene and to stimulate expression of phosphoenolpyruvate (PEP) carboxykinase gene in the liver in vivo. The respective roles, however, of hyperglucagonemia, insulinopenia, and carbohydrate deprivation in the inhibition of L-type pyruvate kinase gene expression during fasting are poorly understood. In addition, the long-term effects of physiological hyperglucagonemia on expression of the two genes are not known.In this study, we investigate the effects of long-term physiological hyperglucagonemia and insulinopenia induced by suckling (which provides a high-fat, low-carbohydrate diet) on expression of the two genes in the liver of normal newborn rats.We show that transcription of the L-type pyruvate kinase gene is inhibited at birth and remains low during the whole suckling period, whereas transcription of the PEP carboxykinase gene is maximal in the neonate, and then decreases despite very high levels of plasma glucagon during suckling.In contrast to the adult, however, in which L-type pyruvate kinase gene expression in the liver is blocked by cAMP and stimulated by carbohydrates, the regulation of L-type pyruvate kinase gene expression in the newborn undergoes a developmental maturation: the inhibitory effect of glucagon is never complete in developing rat liver and the stimulatory effect of glucose could not be detected during suckling, due to either hyperglucagonemia, immaturity of the gene regulatory system, or both.

show abstract

Comparative kinetic studies on the L-type pyruvate kinase from rat liver and the enzyme phosphorylated by cyclic 3′,5′-AMP-stimulated protein kinase

Cited by 94 publications

References 17 publications

Phosphorylation is switch of L-type pyruvate kinase allostery

Phosphorylation is switch of L-type pyruvate kinase allostery

Hormone‐Induced Changes in Pyruvate Kinase

In vivo regulation of glycolytic and gluconeogenic enzyme gene expression in newborn rat liver.

Contact Info

Product

Resources

About