Lecture 17- Gluconeogenesis and the Reciprocal Regulation of Glycolysis & Gluconeogenesis

Describe why we need gluconeogenesis in order to survive & thrive

Gluconeogenesis is the synthesis of glucose from pyruvate and non-carbohydrateprecursors. Gluconeogenesis is especially important during fasting or starvation, as glucose is the primary fuel for the brain and the only fuel for red blood cells

Explain why the three irreversible steps in glycolysis must be bypassed in gluconeogenesis

to bypass, how many enzymes and intermediates are used?

To bypass, 4 new enzymes and 1new intermediate are used

what are the four gluconeogenic reactions that aren't found in glycolysis

Pyruvate Carboxylase

Phosphoenolpyruvate Carboxykinase

Fructose 1,6-Bisphosphatase

Glucose 6-Phosphatase

Pyruvate Carboxylase

Pyruvate Carboxylase - Ligase

The conversion of pyruvate into PEP begins with the formation of oxaloacetate in the mitochondria by pyruvate carboxylase

1. Pyruvate is shuttled from cytoplasm to mitochondrial matrix through the Mitochondrial Pyruvate Carrier (MPC)

2. Pyruvate is carboxylated to the intermediate oxaloacetate by the enzyme pyruvate carboxylase (PC)

3. That oxaloacetate is then reduced to malate by mitochondrial malate dehydrogenase(MDH) and transported into the cytoplasm, where it is re-oxidized to oxaloacetate by cytoplasmic malate dehydrogenase (MDH)

Pyruvate carboxylase uses the vitamin biotin as a coenzyme (activated carrier of CO 2),and its reaction mechanism occurs in 4 steps

Phosphoenolpyruvate Carboxykinase

PEP is then synthesized from oxaloacetate by phosphoenolpyruvate carboxykinase (PEPCK).

Fructose 1,6-Bisphosphatase

Phosphoenolpyruvate is metabolized by the enzymes of glycolysis in the reverse direction until the next irreversible step, the hydrolysis of fructose 1,6-bisphosphate. The enzyme catalyzing this hydrolysis reaction is fructose 1,6-bisphosphatase, an allosteric enzyme

Glucose 6-Phosphatase

The generation of free glucose, which occurs essentially only in liver, is the final step in gluconeogenesis. Glucose 6-phosphate is transported into the lumen of the endoplasmic reticulum. Glucose 6-phosphatase, an integral membrane on the inner surface of the endoplasmic reticulum, catalyzes the formation of glucose from glucose6-phosphate

stoichiometry of gluconeogenesis

2 Pyruvate + 4ATP + 2GTP + 2NADH + 6H2O —> glucose + 4ADP + 2GDP + 6Pi + 2NAD+ + 2H+

Quantify how many ATP equivalents are needed to produce one molecule of glucose from 2 molecules of pyruvate, alanine, glycerol, or lactate

6 ATP

how is glycolysis and gluconeogenesis reciprocally regulated

Gluconeogenesis and glycolysis are coordinately regulated in the liver so that within a cell, one pathway is relatively inactive while the other is highly active

The rationale for reciprocal regulation is that glycolysis will predominate when glucose is abundant, and that gluconeogenesis will be highly active when glucose is scarce

If ATP is needed, glycolysis predominates.

• If glucose is needed, gluconeogenesis is favored

Describe the coordinated regulation of glycolysis and gluconeogenesis in the liver and explain how energy charge and glucose levels affect the coordinated regulation of glycolysis & gluconeogenesis.

The Balance between Glycolysis and Gluconeogenesisin the Liver Is Sensitive to Blood-Glucose Concentration

insulin is secreted by the pancreas in response to high glucose levels. This stimulates glycogen synthesis and glycolysis, as well as fatty acid synthesis and many anabolic pathways.

Glucagon is secreted by the pancreas in response to low glucose levels. This stimulates glycogen breakdown and gluconeogenesis, as well as fatty acid degradation and ketone body synthesis (an alternative fuel derived from fats)

Explain, in detail, how and why fructose 2,6-bisphosphate is the key allosteric regulator of glycolysis and gluconeogenesis

The synthesis and breakdown of fructose 2,6-bisphosphate is dependent on glucose levels

• The kinase that synthesizes fructose 2,6-bisphosphate and the phosphatase that hydrolyzes this molecule are located on the same polypeptide chain, PFK2/FBPase2

.• Such an arrangement is called abifunctional enzyme. The key allosteric regulator of glucose metabolism in liver that responds to blood glucose levels is fructose 2,6-bisphosphate. High fructose 2,6-bisphosphate favor glycolysis, while low fructose 2,6-bisphosphate favor gluconeogenesis. Fructose 2,6-bisphosphate stimulates phosphofructokinase and inhibits fructose 1,6-bisphosphatase.

PFK2/FBPase2

Phosphorylation of the bifunctional enzyme PFK2/FBPase2 activates the phosphataseactivity (FBPase2) and inhibits the kinase activity (PKF2), promoting gluconeogenesis. Dephosphorylation of the bifunctional enzyme activates the kinase activity (PFK2) and inhibits the phosphatase activity (FBPase2), promoting glycolysis.

What is meant by a substrate cycle and metabolic flux? How and why do substrate cycles respond to metabolic flux?

Each bypass in glucogenesis around an irreversible step in glycolysis is called a substrate cycle. The direction of the pathway is reciprocally regulated so that glycolysis and gluconeogenesis don'toccur simultaneously, creating potentially futile cycles

Both reactions of a substrate cycle are not fully active at the same time because of reciprocal allosteric controls.

• It has now been shown though that there can be some detectable activity of opposing pathways at the same time.

• Previously this was considered undesirable and was termed a futile cycle.

• However, it is now believed that substrate cycles can sometimes be biologically important, enhancing metabolic signals.

• A small change in the rates of the two opposing reactions can result in a large change in the net flux.

• Suppose that the rate of conversion of A into B is 100and of B into A is 90, giving an initial net flux of 10

.• Assume that an allosteric effector increases the A → B rate by 20% to 120 and reciprocally decreases the B → A rate by 20% to 72 (net flux of48, an increase of 380%!)

• During intense exercise, flux through glycolysis has been observed to increase 1000-fold!

Explain how interorgan cycles like the Cori cycle and the glucose-alanine cycle function to transfer free energy between the liver and the muscles

Cori cycle(exercise)

Glucose-alanine cycle(protein breakdown)

Muscle and liver display interorgan cooperation in a series of reactions called the Cori cycle and the glucose-alanine cycle. Lactate produced by muscle during contraction is released into the blood. Alanine is released from the breakdown of proteins to amino acids. Liver removes the lactate and alanine, and converts them into glucose, which can be released into the blood.

For the inborn errors of metabolism that we discussed (triose phosphate isomerase deficiency, pyruvate kinase deficiency, and pyruvate carboxylase deficiency),describe how the deficiency of the enzyme affects, at the biochemical level, glycolysis in the red blood cells and/or gluconeogenesis in the liver cells.

Triose phosphate isomerase (TPI) deficiency affects both glycolysis andgluconeogenesis

Pyruvate kinase deficiency affects outflow of glycolysis

Pyruvate carboxylase deficiency affects gluconeogenesis directly and many other processes, too, as oxaloacetate is a key intermediate