Lecture 21: Anabolic Reactions

Gluconeogenesis

• Synthesis of glucose from non-carbohydrate precursors e.g. lactate and alanine via pyruvate conversion

  • Reaction doesn’t occur in all cells → liver and kidney

• Maintains glucose levels in the blood → provides energy to brain and muscle

How do glycolysis and gluconeogenesis handle reversible and irreversible steps?

• Most reactions are reversible.

  • use the same enzymes for these steps.

• 3 Irreversible Steps:

  • Gluconeogenesis uses different enzymes to bypass these steps e.g. Phosphatase in the last step counteracts the kinase used in the first step of glycolysis.

How does gluconeogenesis convert pyruvate back into phosphoenolpyruvate (PEP)?

• Two-Step Process: Requires two enzymes for a more complex conversion than the reverse reaction in glycolysis.

• Enzymes:

  1. Pyruvate Carboxylase: Converts pyruvate to oxaloacetate.

  2. PEP Carboxykinase: Converts oxaloacetate to PEP.

Non-Carbohydrate Pre-Cursors Used In Gluconeogenesis

• Lactate ⇄ pyruvate

• Some amino acids (e.g. alanine) ⇄ pyruvate

  • Removal of NH3 group

• Some amino acids (e.g. aspartate) ⇄ oxaloacetate

• Glycerol (fats) ⇄ dihydroxyacetone phosphate

Overall Equation for Gluconeogenesis

2 Pyruvate + 2 NADH + 4 ATP + 2 GTP + 6 H2O + 2 H+ → Glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi

Net energy investment/gain of gluconeogenesis

• Formation of glucose from pyruvate

• Net Cost: 6 ATP/GTP per glucose molecule.

Pyruvate Decarboxylase (PC)

• An important enzyme that catalyses the metabolically irreversible reaction, that occurs in the mitochondrial matrix

  • addition of CO2 to pyruvate to form oxaloacetate

    Pyruvate + CO2 + ATP → oxaloacetate + ADP + P

• Requires prosthetic group biotin

Regulation of PC

• Allosterically activated by acetyl CoA

• An accumulate of acetyl-CoA signals an abundance of energy and so directs pyruvate to oxaloacetate for gluconeogenesis

  • Liver cells receive a signal

Structure of Pyruvate Decarboxylase

• Tetramer of 4 subunits

• 2 x biotin carboxylase domains

• Site where pyruvate is carboxylate using CO2 to then form oxaloacetate

• Binding of a molecule to its domains induces a conformational change, activating the enzyme

Biotin

• Co-factor

• Described as a prosthetic group when covalently attached to PC via Lys side chain

• Carrier of Activated CO2 - captures CO2 from the atmosphere to form oxaloacetate

  • Carboxylbiotin

How Is Glucose Stored in Plants and Animals

• Starch

• Glycogen - accumulates in the liver

Glycogen

• Polymers of glucose α(1,4), with α(1,6) branching linkage

• Stored as cytosolic granules in the liver and muscles

• Provides a low, but quick source of glucose for a sudden demand of energy

  • Can provide energy in the absence of oxygen - anaerboic activity

1-4 Linkage

• Ensure a linear structre/ polymer

1-6 Linkage

• Introduces branching into the glucose polymer

Glycogenolysis

• Degradation of glycogen to glucose-1,6-phosphate

  • Polymer is depolymerised to release glucose in the blood

• Polymer then converted to Glucose-6-Phosphate by phosphoglucomutase

Uses of Glucose-6-Phosphate

• Glycolysis → ATP energy

• Phosphate removed and then released into the liver/ blood stream

How Does The Liver Raise Blood Glucose Levles

• Glycogen → G1P → G6P → Glucose (glucose 6-phosphatase)

  • glycogen slowly degraded to maintain levels between sleeping and meals

Structure of Glycogen

• Branched polysaccharide chains

  • increase the amount of available ends – allow rapid hydrolysis from these ends to allow the faster release of glucose units for energy

• Has 1-4 and 1-6 glycosidic linkages

Glycogenin

• A protein at the centre of glycogen

  • Acts as a primer for glycogen synthesis

Glycogen Phosphorylase

• Enzyme that catalyses the sequential removal of glucose residues at the then of of glycogen branches chains

• Glycogen (n residues) + Pi ⇄ glucose-1-phosphate + Glucogen (n – 1 residues) from glycogen chain

  • removes 1 molecule of glucose from the end of the chain

Phosphoglucosemutase

• Enzyme that easily converts G1P to G6P

  • Mutase enzyme that changes the position of phosphate

• Phosphorylated serine present in the enzyme to which glucose binds and the phosphate group is then transferred

• This glucose is then phosphorylated to form a bisphosphate and can then change to form G-6-P

Significance of glycogen phosphorylase stopping at 4 residues from a branch point

• Enzyme stops at 4 residues from a branch point, leading to the truncation of glycogen chains, resulting in what is known as limit dextrin.

What 2 Enzymes Remodel The Resulting ‘Limit Dextrin’ In Glycogen Degredation

• Transferase

• a-1,6-glucosidase

Transferase

• Used in limit dextrin remodelling

• Shifts 3 glucose from one outer branch to anohter

  • Further linearises the glycogen chain

α-1,6-Glucosidase

• Used in limit dextrin remodelling

• Glycogen debranching enzyme that removes the branched glucose and leaves an elongated unbranched chain

  • Can be further degraded by glycogen phosphorylase

Steps Involved In Glycogen Sythesis

• Glucose Conversion

  • requires energy

• Isomerisation

Glucose Conversion

• Glucose + ATP → Glucose-6-phosphate (G6P) + ADP

• Enzyme: Glucokinase (in the liver) or Hexokinase (in other tissues)

  • 1st step in glycogen synthesis

Isomerisation

• G6P → Glucose-1-phosphate (G1P)

• Enzyme: Phosphoglucomutase (reversible)

  • Step in glycogen synthesis

How is Glucose-1-phosphate activated and added to the glycogen chain?

• Activation:

  • Glucose-1-phosphate + UTP → UDP-glucose + PPi

  • UDP-glucose is the activated form of glucose

• Incorporation: using glycogen synthase which adds UDP-glucose to the growing glycogen chain, releasing UDP.

UDP-Glucose Formation

• The activated form of glucose for glycogen synthesis

• Catalysed by UDP-glucose pyro phosphorylase

• Liberates the outer two phosphate groups from UTP as PPi (pyrophosphate)

UDP Glucose

• An actived sugar formed by combining glucose-1-phosphate with UTP.

  • makes glucose suitable for addition to the growing glycogen chain

UTP

• Energetically similar to ATP (UTP + ADP ⇌ UDP + ATP)

How much energy (ATP equivalents) are required to incorporate one glucose molecule into glycogen?

• 2 ATP Equivalents:

  • 1 ATP: Glucokinase/Hexokinase reaction (glucose phosphorylation).

  • 1 UTP (equivalent to ATP): Formation of UDP-glucose.

Addition of Glucose To Glycogen Chain

• Free energy required to continue this continuation required UDP free energy

• 1-4 glycosidic bonds form to form the linearised version of glycogen

Glycogen Synthesis: Nulceation

• The need for a starting point or "core" to initiate a process.

• Glycogenin acts as a primer, attaching the first few (10-20) glucose molecules to itself.

  • This creates a core that glycogen synthase can then extend using 1-4 glucosidic bonds 

Linear Growth

• Allows the continuation of Glycogen Synthesis

  • Chain formed from 1-4 glycosidic bond

Glycogen Synthesis: Branch Formation

• A 1,4 glycosidic bond is broken and a block of ~7 glucose is transferred to a more interior site forming a 1,6 linkage

  • glycogen synthase can then enhance this branching

• α1,6-glycosidic linkage assists reaction

• Increases glycogen solubility, and rates of glycogen synthesis and degradation

Glucokinase

• a liver enzyme that catalyses the phosphorylation of glucose to glucose-6- phosphate

  • Diverts glucose when present at high levels to the synthesis of glycogen in the liver

Inhibition of Hexokinase

• Occurs in response to high levels of G6P

Key characteristics of glucokinase.

• Location: Primarily found in the liver.

• Affinity: Lower affinity for glucose (higher Km) compared to hexokinase.

• Function:

  • Active mainly when blood glucose levels are high.

  • Promotes glycogen synthesis (glucose storage).

Key characteristics of hexokinase

• Location: Found in most tissues, including brain and muscle.

• Affinity: High affinity for glucose (lower Km).

• Function:

  • Captures glucose for immediate energy needs.

  • Inhibited by its product, glucose-6-phosphate (G6P), providing feedback regulation.

Glucose Transport

• Glucose enters mammalian cells down a concentration gradient (thermodynamically downhill)

• Passive transport, but can’t cross the membrane as glucose is polar – requires transporters

  • no ATP required – moves down a concentration gradient

GLUT

• a family of passive hexose transporters (up to 12 coding genes present in humans) that facilitate glucose transport – no ATP used

GLUT1 and GLU3

• Present in nearly all mammalian cells, constantly transport glucose into cell under normal conditions, at essentially constant rate

GLUT2

• Present in liver and pancreatic β cells, transports glucose when concentration in blood is high → pancreas detects level → insulin production

  • Pancreatic B-cells have mechanisms to detect levels of glucose in the blood

  • When high – insulin production is triggered

GLUT4

• Insulin dependent transporter present in muscle and fat cells,

  • the numbers increase rapidly in the presence of insulin

• Excess glucose present after meals is stored as glycogen using insulin – accelerates the interconversion

Structure of GLUT1

• Single polypeptide chain, ~500 amino acids

• 12 membrane-spanning α-helices.

• Six of these form a polar channel in the membrane through which glucose travels.

• Both N- and C-termini are on the cytoplasmic side of membrane.

How does insulin trigger increased glucose uptake into cells?

• Insulin (hormone) is secreted by the pancreas when blood glucose levels are high.

• Insulin binds to the insulin receptor (a tyrosine kinase) on target cells.

• This activates a signalling pathway inside the cell, increasing the number of GLUT4 receptors in the membrane

How Does Insluin Promote GLUT4 Translocation

• Insulin binds to its receptor, activating a signalling cascade within the cell.

• The signal triggers vesicles containing GLUT4 to move towards the cell membrane and fuse with it.

• This increases the number of GLUT4 transporters on the cell surface.

• More GLUT4 enables greater glucose uptake from the blood.

• Inside the cell, glucose is phosphorylated and can be used for energy or stored as glycogen in liver and muscles

Insulin

• Peptide hormone - Produced by β-cells of the pancreas

• Two polypeptide chains (a and B) linked by disulphide bonds.

  • The α chain has an intramolecular disulphide bond

• Synthesized as a single-chain precursor, proinsulin, processed by proteases

• Increases the rate of glucose transport into muscle, adipose tissue

  • Stimulates glycogen synthesis in the liver

Regulation of glycolysis and gluconeogenesis.

• Coordinated: When one pathway is active, the other is generally less active.

• Control by Substrate Availability:

  • Glycolysis: Driven by (high) glucose levels.

  • Gluconeogenesis: Driven by the availability of precursors like lactate (and low glucose levels).

Fructose 2,6- Bisphosphate (F2,6BP)

• Synthesis by the phosphorylation of F-6-P by liver PFK2

• Degraded by Fructose bisphosphatase back to F6P

Effect of F2,6BP On Glycolysis

• A STRONG activator of phosphofructokinase – when F26BP is present in high levels, reaction is activated due to the upregulation of PFK

  • increased rate of reaction

Effect of F2,6BP On Gluconeogenesis

• Inhibits F1-6-BP a key enzyme the the metabolic pathway, decreasing rate of reaction

Structure of PFK2/FBPase-2

• 1 polypeptide with 2 domains

  • Kinase domain and phosphate domains→ allows the removal/ addition of phospahte

Reglation of PFK-2/FBPase-2 fUNCTION

• Controlled by the covalent modification of a single Ser residue

  • When phosphorylated - phosphatase activity is prominent; kinase activity is low

  • When unphosphorylated - kinase activity is dominant

Glucagon

• Produced by α-cells of the pancreas in response to low blood glucose

  • Stimulates glycogen degradation

• Maintains blood glucose levels to ensure the brain and muscle function

How does glucagon signalling affect the activity of PFK-2/FBPase-2?

• Glucagon binds to its receptor on liver cells.

• This activates Protein Kinase A (PKA).

• PKA phosphorylates the Ser residue at position 29 in PFK-2/FBPase-2.

• This favors FBPase-2 activity (phosphatase), decreasing F2,6BP levels.

Effect of Low Blood Glucose on Glucagon

• Glucagon levels rise, and insulin levels fall leading to phosphorylation of PFK-2/FBPase-2.

• F2,6BP Decrease This favours the phosphatase activity of the enzyme, decreasing F2,6BP levels.

• Glycolysis is inhibited due to the lack of its activator, F2,6BP.

  • Gluconeogenesis is stimulated, causing the liver to produce glucose

Effect of High Blood Glucose on Glucagon

• Glucagon levels fall and insulin levels rise leading to a dephosphorylation of PFK-2/FBPase-2.

• F2,6BP Increase: This favours the kinase activity of the enzyme, increasing F2,6BP levels.

• Glycolysis is stimulated by high F2,6BP, allowing the body to use excess glucose.

  • Gluconeogenesis is suppressed.

Epinephrine (Adrenaline)

• Produced. by the adrenal glands in response to sudden energy requirements

  • Stimulates the breakdown of glycogen to glucose and its subsequent release in the blood