Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: physicochemical characteristics of the nucleotide binding site, as deduced from fluorescent spectroscopy measurements
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Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] is inactivated by the fluorescent sulfhydryl reagent N-(iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS). The inactivation reaction follows pseudo-first-order kinetics with respect to active enzyme to less than 10% remaining enzyme activity, with a second-order inactivation rate constant of 2.6 min-1 mM-1 at pH 7.5 and 30 degrees C. A stoichiometry of 1.05 mol of reagent incorporated per mole of enzyme subunit was found for the completely inactivated enzyme. Almost complete protection of the enzyme activity and of dansyl label incorporation are afforded by MnADP or MnATP, thus suggesting that 1,5-IAEDANS interacts with an enzyme sulfhydryl group at the nucleotide binding site. The fluorescence decay of the AEDANS attached to the protein shows a single-exponential behavior with a lifetime of 18 ns. A comparison of the fluorescence band position and the fluorescence decay with those of the adduct AEDANS-acetylcysteine indicates a reduced polarity for the microenvironment of the substrate binding site. The quenching of the AEDANS moiety in the protein can be described in terms of a collisional and a static component. The rate constant for the collisional component is much lower than that obtained for the adduct in a medium of reduced polarity. These last results indicate that the AEDANS moiety is considerably shielded from the solvent when it is covalently attached to PEPCK.Keywords:
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Gluconeogenesis
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Gluconeogenesis from (U-<sup>14</sup>C)-lactate occurred in hepatocytes prepared from term fetuses which lack cytosolic phosphoenolpyruvate carboxykinase and was almost completely inhibited by 3-mercaptopicolinate but was relatively insensitive to amino-oxyacetate. 12 h after birth when up to 32% of the total hepatic phosphoenolpyruvate carboxykinase activity was detectable in the cytosol, glucose synthesis was increased 4.4-fold in hepatocytes from fasted neonates and was partially (37%) sensitive to amino-oxyacetate. In livers of fasted 24-hour-old neonates total phosphoenolpyruvate carboxykinase activity was distributed between the mitochondria and the cytosol in the ratio of 60:40. In hepatocytes prepared from such animals, amino-oxyacetate inhibited glucose synthesis by about 56%, suggesting that up to half of the carbon flow from lactate to glucose was via the formation of phosphoenolpyruvate in the mitochondria. These studies indicate an important role for mitochondrial phosphoenolpyruvate carboxykinase in neonatal gluconeogenesis.
Gluconeogenesis
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Abstract An unsymmetrically substituted quaterthiophene with two terminal ferrocenyl groups was prepared as a long π-conjugated system. Electrochemical and spectroscopic studies were carried out to evaluate the electronic and/or electrostatic communication between the two terminals. The one-electron oxidation would occur at one ferrocene moiety specified due to the unsymmetry of the oligothiophene moiety. The one-electron oxidizing species extended into the oligothiophene moiety apparently interacts with the other terminal ferrocene moiety.
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We synthesized [2]rotaxanes with a pyrrole moiety from a [2]rotaxane with a 1,3-diynyl moiety. The conversion of the 1,3-diynyl moiety of the axle component to the pyrrole moiety was accomplished by a Cu-mediated cycloaddition of anilines. The cycloaddition reaction was accelerated when the [2]rotaxane was used as the substrate. The effect of the structure of the pyrrole moiety on the rate of the shuttling was studied.
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Rotaxane
Pyrrole
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Phosphoenolpyruvate Carboxykinase (PEPCK EC 4.1.1.32) catalyzes the conversion of oxaloacetate (OAA) into phosphoenolpyruvate (PEP) in the presence of GTP. In humans, two isoforms of PEPCK are found: cytosolic form (PEPCK‐C, also called PCK1) and mitochondrial form (PEPCK‐M, also called PCK2). PEPCK‐C (PCK1) is a rate‐controlling enzyme in gluconeogenesis. Recent studies found abnormal concentrations of PEPCK in diabetic mice. Overexpression of PEPCK attenuates insulin signaling and decreases hepatic insulin sensitivity in transgenic mice. Our high throughput assay for Phosphoenolpyruvate Carboxykinase activity provides a valuable tool for both mechanistic and therapeutic studies of diabetes as well as avenue for targeting PEPCK activity. The Phosphoenolpyruvate Carboxykinase activity assay is developed by using a set of specific enzymes that convert Phosphoenolpyruvate (PEP) and carbonate into a series of intermediates and hydrogen peroxide, which in turn, reacts with a probe generating a colorimetric signal (OD570 nm). The color intensity is directly proportional to the amount of active Phosphoenolpyruvate Carboxykinase. This assay is simple, sensitive, high‐throughput adaptable and can detect less than 10 μU of Phosphoenolpyruvate Carboxykinase activity per sample. In conclusion, a Phosphoenolpyruvate Carboxykinase (PEPCK) Activity Assay kit is developed. The kit provides easy and robust tool for measuring PEPCK activity in biological and purified samples (BioVision, Inc. Cat. # K359‐100). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
Gluconeogenesis
Phosphoenolpyruvate carboxylase
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Phosphoenolpyruvate partially inhibits the accumulation of Ca(2+) in isolated mung bean (Phaseolus aureus Roxb.) mitochondria. Succinate-supported Ca(2+) uptake is twice as sensitive to phosphoenolpyruvate inhibition as is NADH- or malate/pyruvate-supported Ca(2+) uptake. Pyruvate, atractylate, and ATP, but not ITP, reverse the phosphoenolpyruvate-induced inhibition. Oxaloacetic acid inhibits succinate-supported Ca(2+) uptake completely while partially inhibiting NADH-supported Ca(2+) uptake. The oxaloacetate inhibition of NADH-supported Ca(2+) uptake is greater than that produced by phosphoenolpyruvate. It is suggested that inhibition of Ca(2+) uptake is due to the conversion of phosphoenolpyruvate into oxaloacetate via phosphoenolpyruvate carboxykinase, with oxaloacetate responsible for the actual inhibition of Ca(2+) uptake.
Phosphoenolpyruvate carboxylase
Malate dehydrogenase
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Objective To develop a new series of hydroxyethylamine (HEA) BACE1 inhibitors with nitrogen heterocyclic moiety at N-terminal and find new N-terminal moiety for enhancing BACE1 inhibition activity. Methods New HEA compounds with nitrogen heterocyclic moiety at N-terminal were synthesized and evaluated as BACE1 inhibitors,with (-)-epigallocatechin-3gallate EGCG as a positive control. Results All new compounds were characterized by 1H NMR and ESI-MS. Evaluation of BACE1 inhibition activity showed that the compound Ⅰ6 with indole moiety at N-terminal had BACE1 inhibition activity. Conclusion The results suggested that the indole moiety at N-terminal interact with S2 pocket of BACE1 and be favorable for enhancing BACE1 inhibition activity, Thus, the indole moiety at N-terminal can be used as lead structure for further finding more effient BACE1 inhibitors.
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Phosphoenolpyruvate carboxylase
Bicarbonate
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