Biochemistry I

Overview of Gluconeogenesis,

and Pentose Phosphate Pathway

Dr. Ray

Gluconeogenesis

Cori Cycle

Chapter 16 – part 3

Covered on Exam 3

(includes material from Chapter 20, p.589-590)

Control of Glycolysis

• The flux through the glycolytic pathway must be adjusted in response to conditions both inside and outside the cell. The rate of conversion of glucose into pyruvate is regulated to meet two major cellular needs:

1) the production of ATP, generated by the degradation of glucose

2) the provision of building blocks for synthetic reactions, such as the formation of fatty acids.

1. Which glycolytic enzymes are likely to be sites of control?

The ________________ reactions at steps __________ , which is near equilib in cellular concentrations)

1

2

3

6

7

8

9

10

4

5

In metabolic pathways, enzymes catalyzing essentially irreversible

reactions are potential sites of control.

• Most of the decrease in free energy in glycolysis takes place in the three essentially irreversible steps catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase.

Energetics of Glycolysis

step ? 1

3

10

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Introduction to Metabolism Traits of Metabolism

2. Where does most of the rest of the energy capture occur?

3. If glucose needed to be made, which steps would require the most amount of energy to overcome?

• The energy released in the anaerobic conversion of glucose into two molecules of pyruvate is only a portion of the total energy captured during complete aerobic glucose catabolism.

Gluconeogenesis

1. Why is this metabolic pathway important? because the _______ depends on glucose as its primary fuel and _______________ use only glucose as a fuel

• The daily glucose requirement of the brain in a typical adult human being is about 120 g, which accounts for most of the 160 g of glucose needed daily by the whole body. • The amount of glucose present in body fluids is about 20 g, and that readily available from glycogen, a storage form of glucose, is approximately 190 g.

•Thus, the direct glucose reserves are sufficient to meet glucose needs for about a day. During a longer period of not eating, glucose must be formed from non-carbohydrate sources, through gluconeogenesis.

• The synthesis of glucose from non-carbohydrate precursors (such as pyruvate and lactate) is called gluconeogenesis.

Focus on comparing Gluconeogenesis to Glycolysis

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Red Blood Cells do not have mitochondria, so they do not do aerobic metabolism.

All ATP is made only from glycolysis.

1

2

3

6

7

8

9

10

4

5

Text Fig 16.2

Glycolysis and Gluconeogenesis

• Under typical cellular conditions, most of the decrease in free energy in glycolysis takes place in the three essentially irreversible steps catalyzed by: (1) hexokinase (3) phosphofructokinase (10) pyruvate kinase

• In gluconeogenesis, these three irreversible steps are by-passed by other reactions, in order to overcome the energetic barrier.

• Gluconeogenesis is energetically costly.

Hexokinase

Phosphofructo kinase

Pyruvate kinase http://higheredbcs.wiley.com/legacy/college/boyer/

0471661791/animations/animations.htm

Gluconeogenesis Energetics

DG’ for the formation of pyruvate from glucose (in glycolysis) is about -84 kJ/mol

Gluconeogenesis is not the Reverse of Glycolysis

In gluconeogenesis, the three irreversible steps above are by-passed by other unique reactions, in order to overcome the energetic barrier.

Replace step 10

(with 2 steps)

Replace step 3:

Replace step 1:

10

3

1

Unique Reactions of Gluconeogenesis:

Kinase uses ATP for

phosphate source and

for energy source .

Phosphatases hydrolyze

phosphorylated alcohols

(ROP), which have some

small amount of free

energy of hydrolysis.

Both kinase and

phosphatase reactions

are exergonic.

in glycolysis

(step 3 - kinase)

in gluconeogenesis

(step 8 - phosphatase)

Exergonic hydrolysis catalyzed by Fructose 1,6-bisphosphatase

What is the phosphoryl transfer potential of Pi (inorganic phosphate?

DGo’ = -14.2 kJ/mol

DGo’ = -16.3 kJ/mol

Energetics of Gluconeogenesis

Gluconeogenesis - NOT a reversal of glycolysis

(reactions 3a & 3b) know overview only, NO details

The three exergonic (irreversible)

steps of glycolysis in cell:

are replaced by other favorable

reactions:

(1) Glucose-6-phosphatase

(2) Fructose-1,6-bisphosphatase

Pyruvate Oxaloacetate PEP

(3a) Pyruvate carboxylase

(3b) Phosphoenolpyruvate carboxykinase

(1) Hexokinase

(2) Phosphofructokinase

(3) Pyruvate kinase

phosphorylate

dephosphorylate

Substrate level phosphorylation

Gluconeogenesis vs. Reversal of Glycolysis The stoichiometry of gluconeogenesis is:

Recall that one ATP equivalent changes the equilibrium constant by a

factor of about 108. Hence, the input of four additional high-energy bonds

in Gluconeogenesis changes the equilibrium by a factor of about 1032.

This is a clear example of the coupling of reactions: ATP hydrolysis is used

to power an energetically unfavorable reaction.

In contrast, the stoichiometry for the reversal of glycolysis is:

Gluconeogenesis costs 6 ATP equivalents

Overall, both pathways are highly exergonic (spontaneous)!

Glycolysis

DGo’ = - 84 kJ/mol

produces 2 ATP

• Lactate – formed by lactate dehydrogenase in active skeletal muscle when rate of glycolysis exceed rate of oxidative metabolism (because of insufficient levels of oxygen).

• Amino acids – from proteins in the diet and during starvation from breakdown of proteins in skeletal muscle.

• Glycerol – from hydrolysis of triacylglycerols in fat cells. Glycerol can enter the glycolytic or gluconeogenic pathways through DHAP:

Phosphorylate at C3 Oxidize at C2

Non-carbohydrate Precursors

of Gluconeogenesis

Glycolysis

(1) Hexokinase (- ATP) (3) Phosphofructokinase (- ATP) (10) Pyruvate kinase (+ ATP)

Gluconeogenesis

(11) Glucose-6-phosphatase

(9) Fructose-1,6-bisphosphatase

(1) Pyruvate carboxylase (- ATP) and

(2) Phosphoenolpyruvate carboxykinase (- GTP)

Comparison of Glycolysis & Gluconeogenesis

In most tissues gluconeogenesis stops after formation of glucose-6-phosphate, which can be stored as glycogen. One advantage to ending gluconeogenesis at glucose 6-phosphate is that, unlike free glucose, the molecule cannot diffuse out of the cell. To keep glucose inside the cell, the generation of free glucose is controlled in two ways:

1. Enzyme responsible for the conversion of glucose 6-phosphate into glucose, glucose 6-phosphatase, is regulated.

2. Enzyme is present only in tissues whose metabolic duty is to maintain blood-glucose homeostasis. Tissues that release glucose into the blood are the liver and to lesser extent the kidney.

(7) Phosphoglycerate kinase (+ATP) (5) Phosphoglycerate kinase (-ATP)

happens twice

costs 6 ATP makes 2 ATP

- is used + is made

Gluconeogenesis

Glycolysis and Gluconeogenesis are coordinated, in a tissue-

specific fashion, to ensure that the glucose-dependent energy

needs of ALL cells are met. In a particular cell, when one

pathway is upregulated, the other is downregulated.

Cori Cycle (Figure 16.33) • The main site of gluconegenesis is

in the liver, with a small amount

occurring in the kidneys.

• Gluconeogenesis in these two organs

helps maintain glucose levels in the

blood so that the brain, red blood

cells and muscles can extract

sufficient glucose from blood to

satisfy their energy needs.

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Reciprocal regulation

The Cori Cycle

CORI CYCLE - Lactate formed by active muscle travels through the

blood and is converted into glucose by the liver. This cycle shifts

part of the metabolic burden of active muscle to the liver.

Lactate Dehydrogenase (LDH) Isozymes:

H4, H3M1, H2M2, H1M3, M4

H = heart (higher affinity for lactate) takes

in lactate, oxidizes it to pyruvate, then uses

pyruvate via aerobic metabolism (citric

acid cycle, electron transport chain)

M = skeletal muscle and liver, have lower

affinity for lactate

In the liver, lactate is oxidized to pyruvate by lactate dehydrogenase. This

pyruvate undergoes gluconeogenesis to produce free glucose, which is released

into the blood. The liver restores the level of glucose necessary for active

muscle cells to continue anaerobic glycolysis for immediate energy needs.

Cori Cycle (Figure 16.33)

Carbohydrate Metabolism and the Liver

Workbook, Chapter 16 Self-Test, Q27:

1. Which of the following statements about the Cori cycle and its physiologic consequences are true?

A) It involves the synthesis of glucose in muscle.

B) It involves release of lactate by muscle.

C) It involves lactate synthesis in the liver.

D) It involves ATP synthesis in muscle.

E) It involves release of glucose by the liver.

Gluconeogenesis in liver

Anaerobic glycolysis in muscle

2. How much energy is extracted when one glucose is converted to 2 pyruvate?

3. What is the energy cost of producing one glucose from 2 pyruvate?

Other Metabolic Pathways Associated with Glycolysis

• The entry point of other important dietary sugars into glycolysis varies with the monosaccharide and the organ.

• Common entry points are:

Glycogen

Pentose Phosphate Pathway

PFK

Glucose-6-P

Fructose-6-P

DHAP & GAP

H

C

OH

HHO

OHH

OHH

CH2OH

O H

H

C

OH

HHO

OHH

OHH

CH2

O H

O P

O

O-

O-

CH2OH

O

HHO

OHH

OHH

CH2 O P

O

O-

O-

CH2

O

HHO

OHH

OHH

CH2 O P

O

O-

O-

O P O-

O

O-

F6P FBP

C

OHH

CH2 O P

O

O-

O-

O H

CH2

O

CH2OH

O P O-

O

O-

DHAP

G6PGlucoseGAP

Phospho-rylation

Phospho-rylation

Isomeri-zation

GLYCOGEN FRUCTOSE

PFK

Energy Storage

Biosynthesis

and Growth

hexokinase

NADPH

ribose-5-phosphate

hexo-kinase

Multiple Fates of Glucose-6-Phosphate (G6P)

• 5C Ribose (for DNA/RNA synthesis) and

• NADPH (for reductive biosynthesis of biomolecules)

1. Used as fuel in anaerobic or aerobic glycolysis

2. Converted to free gluocose by liver and released into the blood to raise blood sugar when too low

3. Processed by Pentose Phosphate Pathway, which produces:

Figure 21.3

from Introduction to Chapter 20 (p. 589 – 590)

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First stage of PPP:

Pentose Phosphate Pathway – Needed for Biosynthesis

Anabolism is REDUCTIVE so biosynthesis and growth require the reduced cofactor __________ , which is produced by the pentose phosphate pathway.

CH2OH

O

H OH

H OH

H OH

H

Ribose-5-phosphate

2 Fructose-6-P + glyceraldehyde-3-P

(which feed into glycolysis)

• In PPP glucose-6-phosphate is converted into ribose-5-phosphate (needed for nucleotide biosynthesis to form RNA & DNA), with production of two NADPH for metabolic biosynthesis.

• Excess Ribose-5-P formed is completely converted into the glycolytic intermediates fructose-6-P and glyceraldehyde-3-P.

PHASE 2 (nonixidative)

2 NADP+

2 NADPH

+ CO2

Pentose Phosphate Pathway

Glycogen (energy storage, when energy not needed)

Glucose-6-P

Glycolysis:

• convert glucose into pyruvate

• produce 2 ATP & 2 NADH

Liver exports glucose

Synthetic pathways requiring NADPH:

• Fatty acid biosynthesis

• Cholesterol biosynthesis

• Neurotransmitter biosynthesis

• Nucleotide biosynthesis (and NAD+, FAD, acetyl CoA)

Detoxification processes need NADPH:

• Reduction of oxidized glutathione

• Cytochrome P450 monooxygenases

• The pentose phosphate pathway (occurs in cytosol) meets the need of all organisms for a source of NADPH to use in reductive biosynthesis.

Pyruvate

Gluconeogenesis:

• converts pyruvate into glucose

• utilizes 4 ATP, 2 GTP & 2 NADH

NADPH & ribose-5-P

Metabolism and the Liver

Workbook, Chapter 20 Self-Test, Q13:

Liver synthesizes fatty acids and lipids for export to other tissues. Would you expect the Pentose Phosphate Pathway (PPP) to have a low or a high activity in this organ? Explain.

Biosynthesis of fatty acids requires reducing equivalents in the form of NADPH. In all organs that carry out reductive biosynthesis, the PPP supplies a large portion of the required NADPH.

HIGH

What types of reactions do the following enzymes catalyze?

(A) phosphatase –

(B) kinase –

(C) ATPase –

hydrolyzes phosphate from phosphorylated alcohol so transfers phosphate to H2O

transfers phosphate from ATP to alcohol

hydrolyzes ATP and uses energy released to create a concentration gradient or drive a DG + reaction

Carbohydrate Metabolism and the Liver

Workbook, Chapter 16 Self-Test, Q27:

1. Which of the following statements about the Cori cycle and its physiologic consequences are true?

A) It involves the synthesis of glucose in muscle.

B) It involves release of lactate by muscle.

C) It involves lactate synthesis in the liver.

D) It involves ATP synthesis in muscle.

E) It involves release of glucose by the liver.

Ans: B, D, E

Gluconeogenesis in liver

Anaerobic glycolysis in muscle

2. How much energy is extracted when one glucose is converted to 2 pyruvate?

2 ATP + 2 NADH

3. What is the energy cost of producing one glucose from 2 pyruvate?

4 ATP + 2 GTP + 2 NADH