Gluconeogenesis; Regulation of Glycolysis & Gluconeogenesis Copyright © 1999-2005 by Joyce J. Diwan. All rights reserved. Molecular Biochemistry I

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  • Gluconeogenesis;Regulation of Glycolysis & GluconeogenesisCopyright 1999-2005 by Joyce J. Diwan. All rights reserved.Molecular Biochemistry I

  • Gluconeogenesis occurs mainly in liver.Gluconeogenesis occurs to a more limited extent in the kidney & small intestine under some conditions.Synthesis of glucose from pyruvate utilizes many of the same enzymes as Glycolysis. Three Glycolysis reactions have such a large negative DG that they are essentially irreversible. HexokinasePhosphofructokinasePyruvate Kinase. These steps must be bypassed in Gluconeogenesis.

  • Two Glycolysis reactions are bypassed by simple hydrolysis reactions:Hexokinase (Glycolysis) catalyzes:glucose + ATP glucose-6-phosphate + ADPGlucose-6-Phosphatase (Gluconeogenesis) catalyzes: glucose-6-phosphate + H2O glucose + Pi

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    glucose-6-phosphate glucose

    Glucose-6-phosphatase

    _1061754054.cdx

  • Glucose-6-phosphatase enzyme is embedded in the endoplasmic reticulum (ER) membrane in liver cells.The catalytic site is found to be exposed to the ER lumen. Another subunit may function as a translocase, providing access of substrate to the active site.

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    glucose-6-phosphate glucose

    Glucose-6-phosphatase

    _1061754054.cdx

  • Phosphofructokinase (Glycolysis) catalyzes: fructose-6-P + ATP fructose-1,6-bisP + ADPFructose-1,6-bisphosphatase (Gluconeogenesis) catalyzes: fructose-1,6-bisP + H2O fructose-6-P + Pi

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    fructose-1,6-bisphosphate fructose-6-phosphate

    Fructose-1,6-bisphosphatase

    _1061754154.cdx

  • Bypass of Pyruvate KinasePyruvate Kinase (Glycolysis) catalyzes: phosphoenolpyruvate + ADP pyruvate + ATPFor bypass of the Pyruvate Kinase reaction, cleavage of 2 ~P bonds is required. DG for cleavage of one ~P bond of ATP is insufficient to drive synthesis of phosphoenolpyruvate (PEP). PEP has a higher negative DG of phosphate hydrolysis than ATP.

  • Bypass of Pyruvate Kinase (2 reactions):Pyruvate Carboxylase (Gluconeogenesis) catalyzes:pyruvate + HCO3- + ATP oxaloacetate + ADP + PiPEP Carboxykinase (Gluconeogenesis) catalyzes:oxaloacetate + GTP PEP + GDP + CO2

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    pyruvate oxaloacetate PEP

    Pyruvate Carboxylase PEP Carboxykinase

    _1001272491.cdx

  • Contributing to spontaneity of the 2-step process:Free energy of one ~P bond of ATP is conserved in the carboxylation reaction. Spontaneous decarboxylation contributes to spontaneity of the 2nd reaction. Cleavage of a second ~P bond of GTP also contributes to driving synthesis of PEP.

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    pyruvate oxaloacetate PEP

    Pyruvate Carboxylase PEP Carboxykinase

    _1001272491.cdx

  • Biotin has a 5-C side chain whose terminal carboxyl is in amide linkage to the e-amino group of an enzyme lysine.Pyruvate Carboxylase uses biotin as prosthetic group.

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    biotin

    N subject to carboxylation

    lysine residue

    _1001273843.cdx

    _1061635568.cdx

    _968414373.cdx

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    lysine

    _1123783587.unknown

  • The biotin & lysine side chains form a long swinging arm that allows the biotin ring to swing back & forth between 2 active sites.

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    biotin

    N subject to carboxylation

    lysine residue

    _1001273843.cdx

    _1061635568.cdx

    _968414373.cdx

  • Biotin carboxylation is catalyzed at one active site of Pyruvate Carboxylase. ATP reacts with HCO3- to yield carboxyphosphate. The carboxyl is transferred from this ~P intermediate to N of a ureido group of the biotin ring. Overall: biotin + ATP + HCO3- carboxybiotin + ADP + Pi

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    carboxyphosphate

    _1061750205.unknown

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    carboxybiotin

    lysine residue

    _972241744.cdx

    _1001274410.cdx

    _1061799568.cdx

    _968414373.cdx

  • At the other active site of Pyruvate Carboxylase the activated CO2 is transferred from biotin to pyruvate:carboxybiotin + pyruvate biotin + oxaloacetateView an animation.

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    carboxybiotin

    oxaloacetate

    pyruvate

    biotin

    _1061633622.cdx

    _1061636048.cdx

    _1061636507.cdx

    _1061636588.cdx

    _1061636655.cdx

    _1061636210.cdx

    _1061635428.cdx

    _1061635895.cdx

    _1061634596.cdx

    _972241744.cdx

    _1001274410.cdx

    _968414373.cdx

  • When gluconeogenesis is active in liver, oxaloacetate is diverted to form glucose. Oxaloacetate depletion hinders acetyl CoA entry into Krebs Cycle. The increase in [acetyl CoA] activates Pyruvate Carboxylase to make oxaloacetate.Pyruvate Carboxylase (pyruvate oxaloactate) is allosterically activated by acetyl CoA. [Oxaloacetate] tends to be limiting for Krebs cycle.

    Glucose-6-phosphatase

    glucose-6-P glucose

    Gluconeogenesis Glycolysis

    pyruvate

    fatty acids

    acetyl CoA ketone bodies

    oxaloacetate citrate

    Krebs Cycle

  • If it is desired to bind 2 proteins together for an experiment, biotin may be covalently linked to one protein and avidin to the other.Explore with Chime the biotinyl domain of a carboxylase and the avidin-biotin complex. Avidin, a protein in egg whites with a b barrel structure, tightly binds biotin. Excess consumption of raw eggs can cause nutritional deficiency of biotin.The strong avidin-to-biotin affinity is used by biochemists as a specific "glue."

    avidin

    with bound biotin

  • PEP Carboxykinase catalyzes GTP-dependent oxaloacetate PEP. It is thought to proceed in 2 steps:Oxaloacetate is first decarboxylated to yield a pyruvate enolate anion intermediate. Phosphate transfer from GTP then yields phosphoenolpyruvate (PEP).

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    oxaloacetate PEP

    PEP Carboxykinase Reaction

    _1092082134.unknown

  • In the bacterial enzyme, ATP is Pi donor instead of GTP. In this crystal structure of an E. Coli PEP Carboxykinase, pyruvate is at the active site as an analog of PEP/ oxaloacetate.A metal ion such as Mn++ is required for the PEP Carboxykinase reaction, in addition to a Mg++ ion that binds with the nucleotide substrate at the active site. Mn++ is thought to promote Pi transfer by interacting simultaneously with the enolate oxygen atom and an oxygen atom of the terminal phosphate of GTP or ATP.

    Mg++

    pyruvate

    Mn++

    ATP

    PEP Carboxykinaseactive site ligands PDB 1AQ2

  • The source of pyruvate and oxaloacetate for gluconeogenesis during fasting or carbohydrate starvation is mainly amino acid catabolism. Some amino acids are catabolized to pyruvate, oxaloacetate, or precursors of these. Muscle proteins may break down to supply amino acids. These are transported to liver where they are deaminated and converted to gluconeogenesis inputs. Glycerol, derived from hydrolysis of triacylglycerols in fat cells, is also a significant input to gluconeogenesis.

  • Summary of Gluconeogenesis Pathway:Gluconeogenesis enzyme names in red.Glycolysis enzyme names in blue.

    glyceraldehyde-3-phosphate

    NAD+ + Pi

    NADH + H+

    1,3-bisphosphoglycerate

    ADP

    ATP

    3-phosphoglycerate

    Phosphoglycerate Mutase

    2-phosphoglycerate

    H2O

    phosphoenolpyruvate

    CO2 + GDP

    GTP

    oxaloacetate

    Pi + ADP

    HCO3 + ATP

    pyruvate

    Glyceraldehyde-3-phosphate

    Dehydrogenase

    Phosphoglycerate Kinase

    Enolase

    PEP Carboxykinase

    Pyruvate Carboxylase

    Gluconeogenesis

  • glucose

    Gluconeogenesis

    Pi

    H2O

    glucose-6-phosphate

    Phosphoglucose Isomerase

    fructose-6-phosphate

    Pi

    H2O

    fructose-1,6-bisphosphate

    Aldolase

    glyceraldehyde-3-phosphate + dihydroxyacetone-phosphate

    Triosephosphate

    Isomerase

    (continued)

    Glucose-6-phosphatase

    Fructose-1,6-bisphosphatase

  • Glycolysis & Gluconeogenesis are both spontaneous. If both pathways were simultaneously active in a cell, it would constitute a "futile cycle" that would waste energy. Glycolysis: glucose + 2 NAD+ + 2 ADP + 2 Pi 2 pyruvate + 2 NADH + 2 ATPGluconeogenesis: 2 pyruvate + 2 NADH + 4 ATP + 2 GTP glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 PiQuestions:1. Glycolysis yields how many ~P ? 2. Gluconeogenesis expends how many ~P ? 3. A futile cycle of both pathways would waste how many ~P per cycle ? 264

  • To prevent the waste of a futile cycle, Glycolysis & Gluconeogenesis are reciprocally regulated.Local Control includes reciprocal allosteric regulation by adenine nucleotides. Phosphofructokinase (Glycolysis) is inhibited by ATP and stimulated by AMP.Fructose-1,6-bisphosphatase (Gluconeogenesis) is inhibited by AMP.This insures that