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Carbohydrate Metabolism 1: Pentose Phosphate Pathway, Gluconeogenesis, Reciprocal Regulation. Bioc 460 Spring 2008 - Lecture 33 (Miesfeld). primaquine. The dual function enzyme PFK-2/FBPase-2 controls flux through gluconeogenesis and glycolysis by controlling levels of F-2,6-BP in the cell.

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Carbohydrate Metabolism 1:Pentose Phosphate Pathway, Gluconeogenesis, Reciprocal Regulation

Bioc 460 Spring 2008 - Lecture 33 (Miesfeld)

primaquine

The dual function enzyme PFK-2/FBPase-2 controls flux through gluconeogenesis and glycolysis by controlling levels of F-2,6-BP in the cell

Athletes like Jenna Gresdal rely on the Cori Cycle to maintain glucose levels

Deficiencies in the enzyme glucose-6P dehydrogenase affects 400 million people

slide2

Key Concepts: The Pentose Phosphate Pathway

  • The pentose phosphate pathway takes place entirely within the cytoplasm and is also known as the hexose monophosphate shunt or phosphogluconate pathway.
  • The most important function of the pentose phosphate pathway is to reduce two molecules of NADP+ to NADPH (nicotinamide adenine dinucleotide phosphate) for each glucose-6-phosphate that is oxidatively decarboxylated to ribulose-5-phosphate.
  • NADPH is functionally similar to NAD+ however, NADPH is the primary reductant in the cell, whereas, NAD+ is the primer oxidant. NADPH is critical to maintaining reduced glutathione levels in cells which is required to minimized damage from reactive oxygen species.
  • The pentose phosphate pathway is also responsible for producing ribose-5-phosphate which provides the ribose sugar backbone that anchors the nucleotide base to DNA and RNA polymers.
slide3
We will cover three primary pathways related to carbohydrate metabolism in non-photosynthetic organisms:
  • Pentose phosphate pathway
  • Gluconeogenesis
  • Glycogen metabolism

Metabolism of ribose sugars in the pentose phosphate pathway is used to generate NADPH and to provide the carbohydrate component of nucleotides.

The major sources of carbon in gluconeogenesis are amino acids and glycerol in animals, and glyceraldehyde-3-phosphate (GAP) in plants.

pathway questions
Pathway Questions

1. What does the pentose phosphate pathway accomplish for the cell?

  • The oxidative phase generates NADPH which is required for many biosynthetic pathways and for detoxification of reactive oxygen species.
  • The nonoxidative phase interconverts C3, C4, C5, C6 and C7 monosaccharides to produce ribose-5P for nucleotide synthesis, and also to regenerate glucose-6P to maintain NADPH production by the oxidative phase.
pathway questions5
Pathway Questions

2. What is the overall net reaction of the pentose phosphate pathway when it is utilized to generate the maximum amount of NADPH?

6 Glucose-6P + 12 NADP+ + 12 H2O → 5 Glucose-6P + 12 NADPH + 12 H+ + 6 CO2

pathway questions6
Pathway Questions

3. What are the key enzymes in the pentose phosphate pathway?

Glucose-6P dehydrogenase (G6PD)–This reaction is the commitment step in the pathway and is feedback-inhibited by NADPH.

Transketolase and Transaldolase - together these two enzyme catalyze the reversible "carbon shuffle" reactions of the nonoxidative phase of the pathway.

pathway questions7
Pathway Questions

4. What are examples of the pentose phosphate pathway in real life?

Glucose-6P dehydrogenase G6PD) deficiency is the most common enzyme deficiency in the world and affects over 400 million people. Red blood cells do not produce enough NADPH to protect against reactive oxygen species that are generated by anti-malarial drugs (primaquine) and by compounds in fava beans (vicine).

Malaria-infected Anopheles

mosquito biting a human

A big bowl of fava beans

two phases of the pentose phosphate pathway
Two Phases of the Pentose Phosphate Pathway

The pentose phosphate pathway can be divided into two phases, the oxidative phase, which generates NADPH, and the nonoxidative phase, which regenerates G-6P.

three enzymatic reactions in the oxidative phase

G6PD is the commitment step in the Pentose Phosphate Pathway because 6-Phosphogluconon-d-lactone has no other metabolic fate except to be converted to 6-phosphogluconate.

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Three enzymatic reactions in the oxidative phase
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6

5

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The non-oxidative phase of the PPP

The carbon shuffle reactions of the nonoxidative phase are used to regenerate glucose-6P using the same transketolase and transaldolase enzyme reactions as the Calvin Cycle.

Six glucose-6P (36 carbons) are metabolized to regenerate five glucose-6P (30 carbons).

What happened to 6 carbons?

slide11

Metabolic flux through the Pentose Phosphate Pathway is tightly-regulated

• If increased NADPH is required for biosynthetic pathways.

• If cells need to replenish nucleotide pools.

• If ATP levels in the cell are low.

slide12
Regulation of the G6PD activity controls flux through the glycolytic pathway and pentose phosphate pathways
glucose 6p dehydrogenase deficiency in humans
Glucose-6P dehydrogenase deficiency in humans

The pentose phosphate pathway is responsible for maintaining high levels of NADPH in red blood cells (erythrocytes) for use as a reductant in the glutathione reductase reaction. Glutathione is a tripeptide that has a free sulfhydryl group which functions as an electron donor in a variety of coupled redox reactions in the cell.

slide14

Glucose-6P dehydrogenase deficiency in humans

When erythrocytes are exposed to chemicals that generate high levels of superoxide radicals, GSH is required to reduce these damaging compounds.

The pentose phosphate pathway in erythrocytes normally provides sufficient levels of NADPH to maintain the GSH:GSSG ratio at about 500:1.

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Glucose-6P dehydrogenase deficiency in humans

Primaquine inhibits growth of the malarial parasite in red blood cells by creating a hostile environment. The biochemical basis for this drug-induced illness was found to be lower than normal levels of NADPH due to a G6PD deficiency.

The acute hemolytic anemia seen in individuals with G6PD who are treated with primaquine explains the symptoms of favism. One of the compounds in fava beans is vicine, a toxic glycoside that induces oxidative stress in erythrocytes.

What might explain the observation that cultures with high amounts of fava beans in the diet were associated (in ancient times) with low malaria rates?

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Key Concepts: Gluconeogenesis

  • The importance of gluconeogenesis is to provide glucose for cells from non-carbohydrate precursors.
  • Three steps in glycolysis must be bypassed by gluconeogenic enzymes in order to overcome large G differences.
  • Reciprocal regulation at the PFK-1 (glycolysis) and F-1,6-BPase (gluconeogenesis) is controlled by the allosteric regulator F-2,6-bisphosphate, as well as, energy charge (ATP/AMP), and citrate levels.
  • The Cori Cycle recycles lactate produced in anaerobic muscle cells during exercise by exporting it to the liver where it is converted to pyruvate and used to synthesize glucose by gluconeogenesis.
pathway questions17
Pathway Questions

1. What does gluconeogenesis accomplish for the organism?

The liver and kidney generate glucose from noncarbohydrate sources.

Plants use the gluconeogenic pathway to convert GAP into glucose which is used to make sucrose and starch.

pathway questions18
Pathway Questions

2. What is the overall net reaction of gluconeogenesis?

2 pyruvate + 2NADH + 4ATP + 2GTP + 6H2O →

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

3. What are the key enzymes in gluconeogenesis?

Pyruvate carboxylaseconverts pyruvate to oxaloacetate.Phosphoenolpyruvate carboxykinase (PEPCK) converts oxaloacetate to phosphoenolpyruvate (PEP).Fructose-1,6-bisphosphatase-1 (FBPase-1) catalyzes the dephosphorylation of fructose-1,6BP to form fructose-6P.

Glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6P to form glucose.

application of gluconeogenesis in real life

Pathway Questions

Application of gluconeogenesis in real life

Glucose monitoring devices are based on an assay using the enzyme glucose oxidase which produces gluconate and hydrogen peroxide (H2O2) from glucose.

The level of H2O2 in the sample is detected by an indicator dye that is oxidized in a reaction catalyzed by peroxidase.

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Gluconeogenesis and glycolysis are opposing pathways

Two of the bypass enzymes in gluconeogenesis,fructose-1,6-bisphosphatase-1 (FBPase-1) and glucose-6-phosphatase, simply reverse the reaction

However, 4 extraATP/GTP, and pyruvate carboxylase and phosphoenolypyruvate carboxykinase (PEPCK), are required to catalyze the bypass reaction that converts pyruvate to PEP.

How do you come up with the 4 extra ATP/GTP for gluconeogenesis compared

to glycolysis?

reciprocal regulation of pfk 1 and fbpase 1
Reciprocal regulation of PFK-1 and FBPase-1

The activities of PFK-1 and FBPase-1 are regulated by the allosteric effectors AMP, citrate, and fructose-2,6-bisphosphate (F-2,6-BP), but in a reciprocal manner.

What is the metabolic logic of reciprocal regulation by citrate?

reciprocal regulation of pfk 1 and fbpase 122
Reciprocal regulation of PFK-1 and FBPase-1

In the presence of F-2,6-BP, the affinity of PFK-1 for its substrate fructose-6P is 25 times higher than it is in the absence of F2,6BP.

Looking at the activity curves for FBPase-1 in the presence and absence of F-2,6-BP it can be seen that the affinity of FBPase-1 for its substrate fructose-1,6BP is 15 times lower in the presence of F-2,6-BP.

levels of f 2 6 bp in the cell are controlled by a dual function enzyme called pfk 2 fbpase 2
Levels of F-2,6-BP in the cell are controlled by a dual function enzyme called PFK-2/FBPase-2

The amount of F-2,6-BP in the cell is controlled by the activity of a dual function enzyme containing two catalytic activities, 1) a kinase activity called phosphofructokinase-2 (PFK-2) that phosphorylates fructose-6P to form F-2,6-BP, and 2) a phosphatase activity called fructose-2,6-bisphosphatase (FBPase-2) that dephosphorylates F-2,6-BP to form fructose-6P.

levels of f 2 6 bp in the cell are controlled by a dual function enzyme called pfk 2 fbpase 224

Less F-2,6-BP

and gluconeogenesis

More F-2,6-BP and glycolysis

Levels of F-2,6-BP in the cell are controlled by a dual function enzyme called PFK-2/FBPase-2

When the PFK-2/FBPase-2 dual function enzyme is unphosphorylated, then the PFK-2 activity in the enzyme is stimulated resulting in the net phosphorylation of fructose-6P to produce more F-2,6-BP which stimulates glycolytic flux.

In contrast, when PFK-2/FBPase-2 is phosphorylated, the activity of FBPase-2 is stimulated, leading to less F-2,6-BP, and reduced flux through glycolysis, with a concomitant increase in flux through gluconeogenesis.

levels of f 2 6 bp in the cell are controlled by a dual function enzyme called pfk 2 fbpase 225
Levels of F-2,6-BP in the cell are controlled by a dual function enzyme called PFK-2/FBPase-2

Activation of the glucagon receptor in liver cells results in stimulation of protein kinase A signaling, leading to an increase in gluconeogenesis, whereas, insulin signaling stimulates protein phosphatase-1, leading to an increase in glycolysis .

the cori cycle
The Cori Cycle

The Cori cycle provides a mechanism to convert lactate produced by anaerobic glycolysis in muscle cells to glucose using the gluconeogenic pathway in liver cells. The Cori cycle has an energy cost (4 ATP equivalents), but it is worth it.

These 4 extra ATP/GTP come from the fact that glycolysis yields a net of 2 ATP per glucose, but gluconeogenesis costs a total of 4 ATP and 2 GTP per glucose.

the cori cycle is important for peak performance in athletic competition
The Cori Cycle is important for peak performance in athletic competition

Athletes "warm down" after exercise to enhance circulation so that lactate will be cleared from the muscle and be used in the liver for glucose synthesis via the Cori cycle.