Gluconeogenesis Reactions

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GLUCONEOGENESIS

• Synthesis of glucose from non-carbohydrate precursors
• Occurs mostly in the liver and minor process in kidney
• Kidney produces 10% total glucose during overnight fast

ENZYMES UNIQUE TO GLUCONEOGENESIS

1. Pyruvate carboxylase (mitochondrial matrix)

• Converts pyruvate to oxaloacetate
• Requires 1 ATP, biotin and 1 CO2 (ABC Reaction)

2. Phosphoenol carboxykinase (cytosolic and mitochondrial isozymes)

• Converts oxaloacetate to phosphoenolpyruvate (PEP)
• Consumes 1 GTP and releases 1 CO2

Cytosolic PEPCK

• Used in the malate shuttle: shuttles oxaloacetate from mitochondrion to cytosol via malate
• Pathway dominates when pyruvate is the gluconeogenic substrate
• Mitochondrion: oxaloacetate --> malate (consumes 1 NADH)
• Cytosol: malate --> oxaloacetate (releases 1 NADH)
• Released NADH used in G3P synthesis
• Pyruvate substrate uses cytosolic PEPCK: consumes 2 NADH

Mitochondrial PEPCK

• Pathway dominates when lactate is substrate
• Cytosol: lactate --> pyruvate (releases 1 NADH)
• Released NADH used in G3P synthesis
• Mitochondrial PEPCK: oxaloacetate --> PEP (can cross mitochondrial membranes)
• Lactate substrate uses mitochondrial PEPCK: does NOT consume NADH

PEP converted to glyceraldehyde 3-phosphate in 4 reversible reactions: 1 ATP and 1 NADH consumed (double energy inputs, substrates and products)

Glyceraldehyde 3-phosphate reversibly combines w/ DHAP to form fructose 1,6-bisphosphate

3. Fructose 1,6-bisphosphatase (cytosol)

• Produces fructose 6-phosphate
• Consumes 1 H20 and releases 1 Pi

Fructose 6-phosphate reversibly converts to glucose 6-phosphate

4. Glucose 6-phosphatase (ER membrane-bound)

• Enzyme complex with 4 proteins
  i. Transport protein: G6P from cytosol to ER lumen
  ii. Phosphatase: removes Pi from G6P to form glucose (consumes 1 H2O)
  iii. Transport protein: transports glucose to cytosol
  iv. Transport protein: transports Pi to cytosol

FINAL BOOKKEEPING:
2 Pyruvate + 4ATP + 2GTP + 2NADH + 6H20 --> 1 Glucose + 4ADP + 2GDP + 2NAD+ + 6Pi + 6H+
2 Lactate + 4ATP + 2GTP + 6H2O --> 1 Glucose + 4ADP + 2GDP + 6Pi + 6H+

Lactate substrate lacks net NADH requirement

CLINICAL CORRELATION

Avidin

• Protein in egg whites
• Binds biotin very tightly (biotin required by pyruvate carboxylase)
• Consuming large amounts of raw eggs over an extended period of time can produce biotin deficiency
• Symptoms: CNS problems (lethargy)

Full-Length Text

• Here we will learn the reactions unique to gluconeogenesis in detail.

• To begin, start a table and denote that gluconeogenesis is the synthesis of glucose from non-carbohydrate precursors.
  • It occurs mostly in the liver and is a minor process in the kidney.
  • At the cellular level, it takes place in the mitochondrion, cytosol and endoplasmic reticulum.

• First, draw the enzymes that our unique to gluconeogenesis in order:
  • Pyruvate carboxylase
  • Phosphoenolpyruvate carboxykinase (PEPCK), which has both mitochondrial and cytosolic isozymes.
  • Fructose 1,6 bisphosphatase
  • Glucose-6-phosphatase.

We will learn each of these enzymes and their reactions in detail.

• To begin, let's draw the substrates and products of these four reactions:
  • 2 Pyruvate, a three-carbon molecule.
  • 2 Oxaloacetate, a four-carbon molecule
  • 2 Phosphoenolpyruvate (PEP), a three-carbon molecule
  • Fructose 1,6-bisphosphate, a six-carbon molecule.
  • Fructose 6-phosphate
  • Glucose 6-phosphate
  • Glucose.

Now, let's start with the pyruvate carboxylase reaction.

• Indicate that this is an A-B-C reaction:
  • it consumes ATP,
  • requires biotin as a cofactor, and
  • consumes carbon dioxide.

• 3-carbon pyruvate becomes 4-carbon oxaloacetate with the addition of a carbon dioxide.

• Avidin is a protein in egg whites that binds biotin very tightly.
  • Consuming large amounts of raw eggs over an extended period of time can produce biotin deficiency, the symptoms of which include central nervous problems including lethargy.

Now, for the second reaction, which is catalyzed by PEPCK.

• Show that carbon dioxide is released as 4-carbon oxaloacetate forms 3-carbon PEP.

• Indicate that GTP is consumed.

• Importantly, PEPCK has cytosolic and mitochondrial isozymes, each of which facilitates a different pathway for the first two steps of gluconeogenesis.

Let's illustrate these pathways, now.

• Draw a mitochondrion with two membranes.

• Label the cytosol external to it.

For pathway number one, we will use cytosolic PEPCK.

• Show that pyruvate enters the matrix from the cytosol.
  • For simplicity, we will not double the substrates and products in this diagram.
  • We will also leave out carbon dioxide and all energy requirements except for NADH.

• Show that pyruvate carboxylase converts it to oxaloacetate in the matrix.
  • Oxaloacetate cannot cross the mitochondrial membranes.

• So, indicate that oxaloacetate converts to malate, a reaction that consumes one NADH.
  • Malate can cross the mitochondrial membranes.

• Show that it exits the mitochondrion, and is reconverted to oxaloacetate in the cytosol.

• Indicate that this cytosolic reaction produces one NADH.

• Highlight this cytosolic NADH, we'll return to it shortly.

• Finally, show that the cytosolic PEPCK isozyme catalyzes PEP formation.

Now, for the mitochondrial isozyme and pathway number 2.

• This time, draw lactate in the cytosol.
  • Recall a key function of gluconeogenesis is to clear blood lactate.

• Show that lactate dehydrogenase converts lactate to pyruvate.

• Indicate that this reaction releases one NADH.

• Highlight this cytosolic NADH; again, we'll explain why shortly.

• Indicate that pyruvate enters the mitochondrion, where it is converted to oxaloacetate by pyruvate carboxylase.

• This time, show that mitochondrial PEPCK produces PEP in the matrix.

• Indicate that PEP can cross the mitochondrial membranes and enter the cytosol.

Return to our main diagram and draw multiple arrows to show that five reversible reactions follow.

• Let's take a closer look at these intermediates:
  • 2-phosphoglycerate (2)
  • 3-phosphoglycerate (2).
  • 1,3 bisphosphoglycerate (2).

• Show that this last reaction requires ATP.

• Now, show that 1,3 bisphosphoglycerate reversibly forms glyceraldehyde 3-phosphate.

• Show that this reaction releases a phosphate, and more importantly, consumes NADH.

Remember those cytosolic NADH's that we highlighted?

• They supply this final reaction!

• Highlight this final NADH to emphasize this point!

• Recall we multiply all the substrates and products by 2 because we started with 2 pyruvates.

Now, let's move on to fructose 1,6 bisphosphate.

• Glyceraldehyde 3-phosphate reversibly combines with one DHAP to produce fructose 1,6 bisphosphate.

• Indicate that fructose 1,6-bisphosphatase removes a phosphate from it.

• Indicate that this reaction consumes water.

• Show that fructose 6-phosphate reversibly converts to glucose 6-phosphate.

This brings us to the final reaction.

• Glucose 6-phosphatase removes a phosphate group to produce glucose.

• Show that one molecule of water is consumed.

Let's take a closer look at this reaction.

• Draw an endoplasmic reticulum.

• Draw an enzyme complex with four proteins in its membrane: shade three with the same color.

• Show that the first protein transports glucose-6-phosphate (G6P) from the cytosol into the lumen.
  • Label it a transport protein.

• Show that the second is the phosphatase: it catalyzes the reaction.

• Indicate that the last two proteins transport glucose and phosphate back into the cytosol.
  • Thus, the complex comprises 3 transport proteins and a phosphatase.

Finally, let's account for the energy requirements in gluconeogenesis. We have already multiplied the initial reactions by two (from pyruvate to glyceraldehyde 3-phosphate).

• For our final bookkeeping, let's add the substrates, products and energy inputs together:
  • 2 pyruvate, 4 ATP, 2GTP, 2NADH and 6H2O YIELDS 1 Glucose, 4 ADP, 2 GDP, 2NAD+, 6 phosphate and 6 H+.
  • We did not include all the water molecules consumed in our diagram.

What if we start with lactate instead of pyruvate?

• We lose the NADH energy requirement!

Why?

• Recall that lactate bypasses the malate shuttle by using mitochondrial PEPCK.
• It does not produce malate, and therefore does not need an NADH input.

• Let's write the equation for lactate to emphasize this:
  • 2 lactate, 4ATP, 2GTP and 6H2O YIELDS 1 Glucose, 4ADP, 2GDP, 6 phosphate and 6H+.