Regulation Of Liver Pyruvate Kinase Research Articles

The phosphate moiety of phosphoenolpyruvate does NOT contribute to allosteric regulation of liver pyruvate kinase by fructose-1,6-bisphosphate✝

A linked-function theory for allostery allows for a differentiation between those protein-ligand interactions that contribute the most to ligand binding and those protein-ligand interactions that contribute to the allosteric mechanism. This potential distinction is the basis for analogue studies used to determine which chemical moieties on the allosteric effector contribute to allostery. Although less recognized, the same separation of functions is possible for substrate-enzyme interactions. When evaluating allosteric regulation in human liver pyruvate kinase, the use of a range of monovalent cations (K+, NH4+, Rb+, Cs+, cyclohexylammonium+ and Tris+) altered substrate (phosphoenolpyruvate; PEP) affinity, but maintained similar allosteric responses to the allosteric activator, fructose-1,6-bisphosphate (Fru-1,6-BP). Because crystal structures indicate that the active site monovalent cation interacts directly with the phosphate moiety of the bound PEP substrate, we questioned if the phosphate moiety might contribute to substrate binding, but not to the allosteric mechanism. Here, we demonstrate that the binding of oxalate, a non-phosphorylated substrate/product analogue, is allosterically enhanced by Fru-1,6-BP. That observation is consistent with the concept that the phosphate moiety of PEP is not required for the allosteric function, even though that moiety likely contributes to determining substrate affinity.

Regulation of liver pyruvate kinase and the phosphoenolpyruvate crossroads.

Certain kinetic properties of the pyruvate kinase activity in different tissues of the rat have been studied in fresh extracts with particular attention to their possible value for the regulation of gluconeogenesis and glycolysis.The pyruvate kinase activity in fresh extracts of the gluconeogenic tissues liver and kidney cortex has allosteric properties very marked in near physiological conditions, as follows: (a) co‐operativity in the kinetics with respect to the concentration of phosphoenolpyruvate; (b) strong allosteric inhibition by alanine and ATP, each of which raises the [S]0.5 value and increases the sigmodicity respect to the concentration of phosphoenolpyruvate; and (c) very strong activation by fructose diphosphate, which greatly lowers the [S]0.5 value and shifts the kinetics from markedly sigmoid to hyperbolic, and can fully counteract the inhibitory effects of alanine and ATP.Keeping the extracts of liver and kidney at low temperature (0–2°) leads to desensitization of the pyruvate kinase to the homotropic cooperativity of the phosphoenolpyruvate substrate and to the allosteric inhibition by alanine and ATP and the activation by fructose diphosphate. This cold desensitization is reversible. These facts and their time dependence make it possible to understand previous difficuties to obtain reproducible allosteric effects with the pyruvate kinase of rat liver.The pyruvate kinase of other tissues examined, including heart and adipose tissue, did not exhibit any of the allosteric properties above described for the enzyme of the gluconeogenic tissues.The μmolar range of fructose diphosphate concentrations required for activation of liver pyruvate kinase in the presence of the two physiological inhibitors and within the physiological range of concentrations of phosphoenolpyruvate fits well in order of magnitude with the calculated range of concentrations of free fructose diphosphate prevailing in liver.These results strongly support the hypothesis that an isoenzyme of pyruvate kinase present in liver and kidney has evolved with built‐in specific regulatory mechanisms in order to prevent the diversion of phosphoenolpyruvate from its way to glucose in glucoenogenic situations. An efficient regulation in the reversible switch over from glycolysis to gluconeogenesis seems to be feasible by the interplay of two feedback inhibitors, alanine and ATP, and a forward activator, fructose diphosphate.