Werner Seubert

The Regulation of Pyruvate Kinase

Publisher Summary This chapter discusses the molecular and kinetic properties, and the physiological significance of pyruvate kinase. From a thermodynamic consideration, these enzymes are especially suited for a control of glycolysis, because they catalyze unidirectional steps driving the metabolic flux into one direction. The combined action of the key enzymes of glycolysis and gluconeogenesis at the control points would allow futile cycles, resulting in the hydrolysis of energy-rich phosphate—an energy-wasting process. Additional regulatory properties of the key enzymes of glycolysis should therefore be expected in tissues that are able to carry out both glycolysis and gluconeogenesis, allowing an opposing control of both metabolic processes. Changes of pH in physiological range would, in addition, allow an opposing control of gluconeogenesis and glycolysis at the level of pyruvate kinase and pyruvate carboxylase reactions. Changes of the pH would not only control pyruvate kinase at suboptimal concentrations of PEP and antagonize the negative control of pyruvate kinase by ATP and alanine, but also control in an opposing way, at suboptimal levels of acetyl-CoA pyruvate carboxylase, the enzyme acting in direction of PEP-synthesis. This relationship adds strong support to a physiological significance of both the intracellular pH and acetyl-CoA in the control of glycolysis and gluconeogenesis in liver.

Multiple mitochondrial forms of acetoacetyl-CoA thiolase in rat liver: Possible regulatory role in ketogenesis

Summary Two mitochondrial forms of acetoacetyl-CoA thiolases (A and B) have been obtained from rat liver. A kinetic analysis of thiolytic activities revealed for both enzymes marked inhibition by the substrate acetoacetyl-CoA and negative cooperativity with respect to CoA. In the reverse reaction (condensation) different kinetic properties were evident: The affinity of enzyme A for acetyl-CoA is lowered by acetoacetyl-CoA. A possible allosteric control of enzyme A by acetoacetyl-CoA is deduced from negative cooperativity with respect to binding of acetyl-CoA, depending on the concentrations of acetoacetyl-CoA. On the contrary, enzyme B shows simple Michaelis-Menten kinetics with acetyl-CoA that are not altered by acetoacetyl-CoA. A possible regulatory role of acetoacetyl-CoA thiolase A in ketogenesis is deduced from the above data.

Inactivation and ATP-dependent reactivation of tyrosine aminotransferase in vitro by membrane bound enzymes from rat liver and kidney cortex

Summary In an attempt to learn more about the physiological significance of a membrane bound system responsible for the inactivation and ATP-depending stabilization of renal PEP-carboxykinase in vitro (5, 6), similar studies were extended to hepatic tyrosine amino-transferase (TAT). Like in the case of PEP-carboxykinase the sub-cellular fractions from liver and kidney cortex also inactivate TAT. ATP causes full protection in the presence of the membraneous fractions from liver with an optimum in the mic.rosomal fraction. The inactivation is inhibited by fluoride. Reactivation of TAT by ATP after inactivation favors interconversion of TAT into different forms as the primary cause of the protective effect of ATP. Cyclic AMP and GMP potentiate the protective effect of ATP. The physiological significance of the membrane bound system in control of gluconeogenesis is discussed. Increasing experimental evidence from various laboratories points to a synergistic control of gluconeogenesis in liver and kidney by short term acting hormones via interaction with β- and α-receptors of the cell membrane, respectively (1–4). Both cyclic AMP and GMP have been discussed as possible second messengers of these hormones (4). Inactivation and nucleoside triphosphate dependent stabilization of renal phosphoenolpyruvate (PEP) carboxykinase in vitro by a membrane bound enzyme system from liver and kidney (5, 6) locate one of the possible regulatory attacks of the above hormones at the pyruvate/PEP level. In view of numerous reports on a cyclic AMP mediated control of tyrosine aminotransferase (TAT) and PEP-carboxykinase in liver, and of gluconeogenesis in both kidney and liver, the specifity studies with the membrane bound enzyme systems from both organs were extended to TAT from liver. The present study on TAT represents a first experimental support for a broad specifity of the membrane bound system for at least the glucogenic enzymes.

Mitochondriale Fettsäure-Synthese und pathologische Ketonkörperbildung

Malonyl-CoA independent fa t ty acid synthesis in mitochondria could be characterized as a reversal of the fi-oxidation involving thiolase, fi-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase and a new enzyme, enoyl-CoA reductase. Both NADH and NADPH function as hydrogen donors for the synthesizing procesS. A new concept of ketone body production is developed. It involves a limitation of fa t ty acid oxidation at the level of the thioclastic cleavage of acetoacetyl-CoA, thus favoring conversion to free acetoacetate independent of the size of the intracellular acetyl-CoA pool.

Control of pyruvate carboxylase in alloxan diabetic rats

Abstract In order to contribute to a clarification of the contradictory reports from different laboratories concerning the control of pyruvate carboxylase in diabetes, the levels of this enzyme were determined in rat liver at different stadiums of alloxan diabetes. In agreement with the former reports unchanged and elevated levels of pyruvate carboxylase were found at the early and late stadium of diabetes, respectively. The implications of this finding concerning a possible role of glucocorticoids in the control of gluconeogenesis in diabetes, is discussed.

In vitro stimulation of gluconeogenesis from pyruvate and of 14CO2-fixation by dexamethasone phosphate and vasopressin in the kidney cortex of adrenalectomized rats

Abstract Dexamethasone phosphate and vasopressin stimulate glucose synthesis and 14CO2-fixation of adrenalectomized rat kidney cortex explants in vitro . 3′,5′-cyclic-AMP demonstrates similar effects as vasopressin. In contrast to dexamethasone phosphate, vasopressin and 3′,5′-cyclic-AMP act without any lag phase. Their stimulatory effects are additive to that of the adrenal steroid. All hormonal effects are suppressed by cycloheximide. The implications of these results concerning different mechanisms of action for the adrenal steroid and vasopressin (or 3′,5′-cyclic-AMP) will be discussed.

Purification and Properties of Pyruvate Carboxylase from Rat Liver

Summary Pyruvate carboxylase from rat liver was purified to almost homogenity. The 180-fold purified preparations have a specific activity of 22 units/mg protein. The enzyme from rat liver shows similar characteristics as already described for the enzyme from chicken liver. It differs from the latter enzyme in its response to acetyl-CoA, an allosteric activator of pyruvate carboxylase. Like isocitrate in the case of mammalian acetyl-CoA carboxylase, acetyl-CoA stimulates the transfer of enzyme bound CO2 (“active CO2”) to the acceptor (eq. 2) catalyzed by pyruvate carboxylase: The activation curve shows a sigmoid shape in a concentration range between 0.5 to 2 × 10-4 M acetyl-CoA. The stimulatory effect of acetyl-CoA in the C14-pyruvate-oxalacetate exchange assay (eq. 2) is associated with an increased maximum velocity and has no apparent effect on the Km-value for pyruvate. Incubation of purified pyruvate carboxylase under various conditions (ATP, Mg++, KHCO3, dilutions etc.) results in extreme differences of the catalytic properties when assayed with the conventional overall assay (eq. 1 and 2) and the C14-pyruvate-oxalacetate exchange assay (eq. 2). The implication of these findings concerning different forms of the enzyme from rat liver will be discussed.