Gluconeogenesis

Gluconeogenesis

Some tissues, such as • brain, RBCs, kidney medulla, testes, embrionic

tissues and exercising muscle require a continuing supply of glucose as a metabolic energy.

• The human brain requires over 120 gm of glucose per day. Mammalian cells make glucose from simpler precursors. Liver glycogen can meet these needs only for 10 to 18 hours without dietary carbohydrate.

During a prolonged fast,

Hepatic liver stores are depleted, glc is formed from other molecules such as

• Lactate• Pyruvate• Glycerol• Alpha keto acids

The formation of glc from nonhexose precursors is called gluconeogenesis (formation of new sugar).

Glycolysis and Gluconeogenesis in the liver

Gluconeogenesis

Pyruvate precursors

The direct Glc reserves are sufficient to meet Glc needs for about a day!

Gluconeogenic pathway makes Glc from pyruvate precursors.

Triacyl glycerol-------> Glycerol + Fatty acids Glycerol is a precursor of glc, glycerol enters glycolytic

pathway as dihydroxyacetone phosphate.

Gluconeogenesis is NOT a reversal of glycolysis

Several reactions MUST differ because of the irreversible steps.

• HK (hexokinase)• PFK (phosphofructokinase)• PK (pyruvate kinase)

Let’s make Glc from pyruvate

• 1. Carboxylation of pyruvate– Pyruvate+CO2+ATP+H2O-----> OA+ADP+Pi+2H

• Enzyme:Pyruvate carboxylase

• OA+GTP----->PEP+GDP+CO2

• Enzyme: PEP-carboxykinase

Domain structure of pyruvate carboxylaseATP grasp: activate bicarbonate ions and transfers CO2 to thebiotin domain. From there CO2 is transferred to pyruvate..

Carboxylation of pyruvate

Pyruvate carboxylase contains BIOTIN, which is covalently bound to the enzyme through lysine

• Enzyme + CO2 + ATP-----> Carboxybiotin-enzyme +ADP +Pi

• Carboxybiotin-enzyme + pyruvate------->OA + Enzyme

– BIOTIN carries CO2...

Biotin is covalently attached group

Biotin serves as a carrier for activated CO2.

-amino group and carboxylate group of biotin are linked. CO2 is found mainly as HCO3 in our system.

When Acetyl CoA is high then biotin is carboxylated.

The activated carboxyl group is transferred from carboxybiotin to pyruvate to form oxaloacetate.

2. Transport of OA to the cytoplasm

• Pyuvate carboxylase is a mitochondrial enzyme, whereas the other enzymes in gluconeogenesis are cytoplasmic.

• OA should be transported to the cytoplasm.• How? • It is reduced to MALATE first then

transferred to the cytoplasm.• In the cytoplasm, it is reoxidized to OA.

3. Decarboxylation of cytoplasmic OA

OA is decarboxylated and P-lated by PEP carboxykinase in the cytosol (PEP is made then!)

The overall reaction catalyzed by the combined action of pyruvate carboxylase and PEP carboxykinase provides a pathway from – Pyruvate-------->PEP

– Therefore, once PEP is formed, it enters the reversed reactions of glycolysis until it reaches F-1,6 Bisphosphate!

4. Dephosphorylation of F-1,6BP

Fructose 1,6-bisphosphate + H2O------> F-6-P + Pi

• Enzyme: Fructose1,6-bisphosphatase

• This enzyme plays an important role in regulation.

• It is inhibited by F 2,6 BisP, an allosteric modifier whose concentration is influenced by the levels of circulating glucagon.

• This enzyme is found in liver and kidney.

5. Generation of free Glc

Dephospharylation of Glc 6-P Glc 6-P + H2O------> D-Glc + Pi

• Enzyme: Glc 6-phosphatase• It is found in liver and kidney but not in

muscle and brain.• Thus, muscle and brain cannot make Glc by

gluconeogenesis• Type I glycogen storage disease results from

an inherited deficiency of glc 6-phosphatase.

Freeing Glc

The final step, freeing Glc takes place in ER lumen where it is hydrolyzed to Glc by Glc 6-Phosphatase which is a membrane bound enzyme.

Calcium binding protein (SP) is necessary for phosphatase activity.

Glc and Pi are shuttled back to the cytosol by a pair of transporters.

The glucose transporter in the ER membrane is like those found in the plasma membrane.

Gluconeogenesis is energetically costly!

The stoichiometry of gluconeogenesis is:

2pruvate + 4ATP + 2GTP + 2NADH + 6H2O-------->Glc

4ADP + 2GDP + 6Pi + 2NAD+ + 2H+

In contrast, the stoichiometry of reversal of glycolysis is:

2 pyruvate + 2ATP + 2NADH + 2H2O------->

Glc + 2ADP + 2Pi + 2NAD+

• The difference is 4ATP, this is needed to turn

energetically unfavorable process to a favorable one!

Gluconeogenesis and glycolysis are reciprocally regulated

Both glycolysis and gluconeogenesis are highly exorgonic under cellular conditions so there is no thermodynamic barrier.

But, amounts and activities of the distinctive enzymes of each pathway are controlled so that both pathways are not highly active at the same time.

Substrate cycles

F-6-P------->F 1,6BisP <--------- A pair of reactions such as the above one is called “substrate

cycle” There is also some cycling in irreversible reactions. “Imperfection” in metabolism? They are sometimes referred as “futile cycles”

• Futile cycles amplify metabolic signals!

• The other potential biological role of substrate cycles is the generation of heat produced by the hydrolysis of ATP.

Lactate and alanine formed by contracting muscle are used by other organs Lactate is a dead end in metabolism. Lactate should be converted to pyruvate. The plasma membranes of most cells are highly permeable

to lactate and pyruvate, therefore they easily diffuse to go to liver!

Excess lactate enters the liver and is converted pyruvate first then glucose.

• Thus, the liver restores the level of glucose necessary for active muscle cells, which derive ATP from the glycolytic conversion of glucose into lactate. Contracting skeletal muscle supplies lactate to the liver, which uses it to make glucose.

• These reactions constitute CORI CYCLE.

LDH enzyme

Lactate-------> Pyruvate by LDH (lactate dehydrogenase).

The interconversion of pyruvate and lactate are done by different subunits of LDH. LDH is a tetramer.

H---> in he heart M---> in the muscle

The Cori Cycle

Cooperation between glycolysis and gluconeogenesis

REGULATION

1. Control point: Pyruvate carboxylase, Acetyl CoA is a + allosteric modulator for pyruvate carboxylase enzyme.– Glc is made from pyruvate when there is a lot Acetyl CoA(more

Acetyl CoA than TCA cycle can handle)

– Acetyl CoA inhibits pyruvate dehydrogenase enzyme but stimulates pyruvate carboxylase.

2. Control point: F 1,6 bisphoshatase reaction

3.Control point: Hormonal control: F-2,6 bisphosphate

Two alternative fates of pyruvate

2nd control point

Hormonal Control

The special role of liver to maintain constant blood glucose level requires additional control mechanisms.

When blood glucose decreases, glycogen increases and glucose is released.

This hormonal regulation in liver is mediated by fructose-2,6-bisphosphate, which is a allosteric effector for PFK-1, and F-1,6-bisphosphate

Role of F2,6BP in regulation of Glycolysis and Gluconeogenesis

What is F-2,6-BP?

It is structurally related to F-1,6-BP. It is not an intermediate. It is a “regulator” F-2,6-BP activates PFK-1 and glycolysis. FBPase and PFK-2 are part of the same enzyme! An increase in glucagon (during starvation) leads to a

decrease in F-2,6-BP overall which goes to a decrease in glycolysis, an increase in glucone ogenesis

A decrease in glucagon (after carbohydrate rich diet) leads to an increase in F-2,6-BP and an increase in glycolysis.

Therefore, F-2,6-BP acts as an intracellular signal indicating “glucose abundant”.