Studies on the Kinetic Effects of Adenosine‐3′: 5′‐MonophosphateDependent Phosphorylation of Purified Pig‐Liver Pyruvate Kinase Type L

Abstract

The effect of cyclic-AMP-dependent phosphorylation on the activity of isolated pig liver pyruvate kinase was studied. It was found that the major kinetic effect of the phosphorylation was to reduce the affinity for the substrate phosphoenolpyruvate, ,5 for this substrate increasing from 0.3 to 0.9 mM upon phosphorylation. The cooperative effect with phosphoenolpyruvate was enhanced, the Hill constant nH increasing concomitantly from 1.1 to 1.5. V was unaltered. The change in activity occurred in parallel with the phosphate incorporation, except during the initial part of the reaction, when inactivation was correspondingly slower. The affinity for the second substrate ADP was unchanged, with an apparent K,,, of 0.3 mM at saturating concentration of phosphoenolpyruvate. Likewise, the requirement for potassium was unaffected, whereas the phosphoenzyme required a higher concentration of magnesium ions for maximal activity, compared with the control enzyme. The inhibitory effect of the phosphorylation was counteracted by positive effectors, fructose 1,6-bisphosphate in micromolar concentrations completely activated the phosphoenzyme, resulting in an enzyme with properties similar to the fructose I ,6-bisphosphateactivated unphosphorylated enzyme, with KO ,5 for phosphoenolpyruvate about 0.025 mM and with a Hill constant of 1 .l. Hydrogen ions were also effective in activating the phosphoenzyme. Thus, when pH was lowered from 8 to 6.5 the inhibition due to phosphorylation was abolished. The phosphoenzyme was sensitive to further inhibition by negative effectors such as ATP and alanine. 2 mM ATP increased I& for phosphoenolpyruvate to 1.5mM and nH to 2.3. The corresponding values with alanine were 1.3 mM and 3.9. Phosphorylation is thought to be an additional mechanism of inhibition of the enzyme under gluconeogenetic conditions.

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

“…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).…”

“…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).…”

Regulation in vitro and in vivo of Adenosine 3′:5′‐monophosphate‐Dependent Inactivation of Rat‐Liver Pyruvate Kinase Type L

“…Treatment of isolated liver preparations with cyclic AMP or glucagon causes modifications of the kinetic properties of pyruvate kinase [3 -61 that are similar to those found in vitro after phosphorylation of purified liver pyruvate kinase [7,8]. The fact that the inactivation of pyruvate kinase by glucagon, cyclic AMP and epinephrine in isolated hepatocytes is parallel with a stimulation of gluconeogenesis, strongly suggests that phosphorylation of pyruvate kinase plays a…”

Phosphorylation of Human Erythrocyte Pyruvate Kinase by Soluble Cyclic‐AMP‐Dependend Protein Kinases

“…Ljungstrom et al were the first to report that rat liver L-type pyruvate kinase can be phosphorylated by an adenosine-3',5'-phosphatedependent (cyclic-AMP-dependent) protein kinase [l]. Phosphorylation of pig-liver L-type enzyme has also been reported [2,3]. Glucagon induces phosphorylation of liver L-type pyruvate kinase in vivo through a cyclic-AMP-dependent mechanism [4-61.…”

“…Glucagon induces phosphorylation of liver L-type pyruvate kinase in vivo through a cyclic-AMP-dependent mechanism [4-61. This phosphorylation of L-type pyruvate kinase causes important changes in the kinetics of the enzyme: decreased affinity for phosphoenolpyruvate, and increased inhibition by the allosteric effectors ATP and alanine [2,. These kinetic modifications are expected to play an important physiological role in the dynamic balance between glycolysis and gluconeogenesis by blocking the pyruvate kinase reaction when gluconeogenesis is the predominant pathway, thus impairing a futile cycle between phosphoenolpyruvate and pyruvate.…”