MLS 064: Biochemistry

Module #6 Student Activity Sheet

Name: Class number:

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_____ Date:
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Lesson title: Gluconeogenesis and Pentose-Phosphate Materials:

Pathway Book, Pen and Notebook
Learning Targets: References:
At the end of the module, students will be able to:
1. Define gluconeogenesis and Pentose-phosphate Ubalde, Biochemistry for Allied Health
pathway Sciences: Edric Publishing, 2019
2. Describe the pathway of gluconeogenesis and its
control.
3. Describe the Cori cycle.
4. Identify the difference between oxidative and
nonoxidative phases of PPP.
5. Enumerate the different functions or importance of PPP.

A. LESSON PREVIEW/REVIEW

a. What are carbohydrates?

b. Classifications of carbohydrates?
c. What are epimers, anomers and enantiomers?

B. MAIN LESSON

The students will study and read their book about this lesson.

GLUCONEOGENESIS

In order to provide glucose for vital functions such as the metabolism of RBC's and the CNS during
periods of fasting (greater than about 8 hours after food absorption in humans), the body needs a way to
synthesis glucose from precursors such as pyruvate and amino acids. This process is referred to as
gluconeogenesis. It occurs in the liver and in kidney. Most of Glycolysis can be used in this process since most
glycolytic enzymes are operating at equilibrium. However three irreversible enzymes must be bypassed in
gluconeogenesis vs. glycolysis: Hexokinase, Phosphofructokinase, and Pyruvate kinase. Phosphofructokinase,
and/or hexokinase must also be bypassed in converting other hexoses to glucose.

Generally, the gluconeogenesis is the reverse of glycolysis. The following three steps are circumvented
for this event to occur:

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 MLS 064: Biochemistry
Module #6 Student Activity Sheet

Name: Class number:

____________________________________________________________ _______
_____ Date:
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a. Pyruvate and Phosphoenolpyruvate

Reversal of the reaction catalyzed by pyruvate kinase in glycolysis involves two endothermic reactions.
Mitochondrial pyruvate carboxylase catalyzes the carboxylation of pyruvate to oxaloacetate, an ATP-requiring
reaction in which the vitamin biotin is the coenzyme. Biotin binds CO from bicarbonate as carboxybiotin prior to
2

the addition of the CO to pyruvate. The resultant oxaloacetate is reduced to malate, exported from the
2

mitochondrion into the cytosol and there oxidized back to oxaloacetate. A second enzyme, phosphoenolpyruvate
carboxykinase, catalyzes the decarboxylation and phosphorylation of oxaloacetate to phosphoenolpyruvate
using GTP as the phosphate donor. In liver and kidney, the reaction of succinate thiokinase in the citric acid
cycle produces GTP (rather than ATP as in other tissues), and this GTP is used for the reaction of
phosphoenolpyruvate carboxykinase, thus providing a link between citric acid cycle activity and
gluconeogenesis, to prevent excessive removal of oxaloacetate for gluconeogenesis, which would impair citric
acid cycle activity.

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 MLS 064: Biochemistry
Module #6 Student Activity Sheet

Name: Class number:

____________________________________________________________ _______
_____ Date:
Section: ____________ Schedule: ________________
________________________________________

b. Fructose 1,6-Bisphosphate & Fructose-6-Phosphate

The conversion of fructose 1, 6-bisphosphate to fructose-6- phosphate, for the reversal of glycolysis, is
catalyzed by fructose 1, 6-bisphosphatase. Its presence determines whether a tissue is capable of synthesizing
glucose (or glycogen) not only from pyruvate, but also from triose phosphates. It is present in liver, kidney, and
skeletal muscle, but is probably absent from heart and smooth muscle.

c. Glucose-6-Phosphate & Glucose

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 MLS 064: Biochemistry
Module #6 Student Activity Sheet

Name: Class number:

____________________________________________________________ _______
_____ Date:
Section: ____________ Schedule: ________________
________________________________________

The conversion of glucose-6-phosphate to glucose is catalyzed by glucose-6-

phosphatase. It is present in liver and kidney, but absent from muscle, which, therefore, cannot export glucose
into the bloodstream. Students should remember that glucose-6-phosphate is also involved in glycogenolysis,
which will be discussed in future modules.

Regulation of Gluconeogenesis

a. Under fasting conditions, glucagon is elevated and stimulates gluconeogenesis. Because of the changes
in the activity of certain enzymes, futile cycles are prevented from occurring, and the overall flow of carbon
is from pyruvate to glucose.
b. A futile cycle is the continuous recycling of substrates and products with the net consumption of energy
and no significant change in substrate levels. This is also known as substrate cycling, as the same
substrate is continuously synthesized and degraded (recycled).
 Pyruvate dehydrogenase
 Decreased insulin and increased glucagon stimulate the release of fatty acids from adipose
tissue.
 Fatty acids travel to the liver and are oxidized, producing acetyl-CoA, NADH, and ATP, which
cause inactivation of pyruvate dehydrogenase.
 Because pyruvate dehydrogenase is relatively inactive, pyruvate is converted to oxaloacetate,
not to acetyl-CoA.
 Pyruvate carboxylase
 Acetyl-CoA (which is generated from fatty acid oxidation within the mitochondria) activates
pyruvate carboxylase, which converts pyruvate to oxaloacetate.
 Note that pyruvate carboxylase is active in both the fed and fasting states.
 Phosphoenolpyruvate carboxykinase (PEPCK)
 PEPCK is an inducible enzyme.
 Increased production of PEPCK mRNA leads to increased translation, resulting in higher
PEPCK levels in the cell.
 Pyruvate kinase
 Glucagon, via cAMP and protein kinase A, causes pyruvate kinase to be phosphorylated and
inactivated.
 Because pyruvate kinase is relatively inactive, phosphoenolpyruvate formed from
oxaloacetate is not reconverted to pyruvate but in a series of steps, forms fructose-1, 6-
bisphosphate, which is converted to fructose-6-phosphate.
 Phosphofructokinase 1
 PFK1 is relatively inactive because the concentrations of its activators, AMP and F-2, 6-P, are
low and its inhibitor, ATP, is relatively high.
 Fructose 1,6-bisphosphatase
 The level of F-2, 6-P, an inhibitor of fructose 1, 6-bisphosphatase, is low during fasting.
Therefore, fructose 1, 6-bisphosphatase is more active.

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 MLS 064: Biochemistry
Module #6 Student Activity Sheet

Name: Class number:

____________________________________________________________ _______
_____ Date:
Section: ____________ Schedule: ________________
________________________________________

 Fructose 1, 6-bisphosphatase is also induced in the fasting state.

 Glucokinase
 Glucokinase is relatively inactive because it has a high Km for glucose, and under conditions
that favor gluconeogenesis, the glucose concentration is low. Therefore, free glucose is not
reconverted to glucose-6-phosphate.

Precursors for gluconeogenesis

Lactate, amino acids, and glycerol are the major precursors for gluconeogenesis in humans.

1. Lactate is oxidized by NAD in a reaction catalyzed by LDH to form pyruvate, which can be converted to
+

glucose. The sources of lactate include red blood cells and exercising muscle.
2. Amino acids for gluconeogenesis come from degradation of muscle protein.
a. Amino acids are released directly into the blood from muscle, or carbons from amino acids are converted to
alanine and glutamine and released.
 Alanine is also formed by transamination of pyruvate that is derived by the oxidation of glucose.
 Glutamine is converted to alanine by tissues such as gut and kidney.
b. Amino acids travel to the liver and provide carbon for gluconeogenesis. Quantitatively, alanine is the major
gluconeogenic amino acid.
c. Amino acid nitrogen is converted to urea.
3. Glycerol, which is derived from adipose triacylglycerols, reacts with ATP to form glycerol-3-phosphate, which
is oxidized to DHAP and converted to glucose.

Cori cycle

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 MLS 064: Biochemistry
Module #6 Student Activity Sheet

Name: Class number:

____________________________________________________________ _______
_____ Date:
Section: ____________ Schedule: ________________
________________________________________

As discussed, glucose is formed from two groups of compounds that undergo gluconeogenesis (1) those
which involve a direct net conversion to glucose, including most amino acids and propionate; and (2) those which
are the products of the metabolism of glucose in tissues. Thus lactate, formed by glycolysis in skeletal muscle
and erythrocytes, is transported to the liver and kidney where it reforms glucose, which again becomes available
via the circulation for oxidation in the tissues. This process is known as the Cori cycle, or the lactic acid cycle.

In the fasting state, there is a considerable output of alanine from skeletal muscle, far in excess of the
amount in the muscle proteins that are being catabolized. It is formed by transamination of pyruvate produced
by glycolysis of muscle glycogen, and is exported to the liver, where, after transamination back to pyruvate, it is
a substrate for gluconeogenesis. This glucose-alanine cycle (see photo) thus provides an indirect way of utilizing
muscle glycogen to maintain blood glucose in the fasting state. The ATP required for the hepatic synthesis of
glucose from pyruvate is derived from the oxidation of fatty acids.

The pentose-phosphate pathway is an alternative route for the metabolism of glucose. It does not lead to
formation of ATP. The pentose phosphate pathway (also called the phosphogluconate pathway and the hexose
monophosphate shunt) has three major functions:

a. The formation of NADPH for synthesis of fatty acids and steroids.

b. Maintenance of reduced glutathione for anti-oxidant activity.
c. The synthesis of ribose for nucleotide and nucleic acid formation.

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 MLS 064: Biochemistry
Module #6 Student Activity Sheet

Name: Class number:

____________________________________________________________ _______
_____ Date:
Section: ____________ Schedule: ________________
________________________________________

Glucose, fructose and galactose are the main hexoses absorbed from the gastrointestinal tract,
derived from dietary starch, sucrose and lactose, respectively. Fructose and galactose can be converted to
glucose, mainly in the liver. Genetic deficiency of glucose-6-phosphate dehydrogenase, the first enzyme of the
PPP, causes acute hemolysis or red blood cells, resulting in hemolytic anemia.

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 MLS 064: Biochemistry
Module #6 Student Activity Sheet

Name: Class number:

____________________________________________________________ _______
_____ Date:
Section: ____________ Schedule: ________________
________________________________________

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 MLS 064: Biochemistry
Module #6 Student Activity Sheet

Name: Class number:

____________________________________________________________ _______
_____ Date:
Section: ____________ Schedule: ________________
________________________________________

 The photo above shows the pentose-phosphate pathway and its connections with the pathway of
glycolysis.

The pentose-phosphate pathway is a more complex pathway than glycolysis. Three molecules of glucose-6-
phosphate give rise to three molecules of carbon dioxide and three 5-carbon sugars. These are rearranged to
regenerate two molecules of glucose 6-phosphate and one molecule of the glycolytic intermediate,
glyceraldehyde-3-phosphate. Since two molecules of glyceraldehyde-3-phopshate can regenerate glucose-6-
phosphate, the pathway can account for the complete oxidation of glucose.
The pentose phosphate pathway consists of two phases: (1) the oxidative generation of NADPH
and (2) the nonoxidative interconversion of sugars. In the oxidative phase, NADPH is generated when glucose
6-phosphate is oxidized to ribulose 5-phosphate:

Ribulose 5-phospate is subsequently converted into ribose 5-phosphate, which is a precursor to

RNA and DNA as well as to ATP, NADH, FAD, and coenzyme A. In the nonoxidative phase, the pathway
catalyzes the interconversion of three-, four-, five-, six-, and seven-carbon sugars in a series of nonoxidative
reactions. Excess five-carbon sugars may be converted into intermediates of the glycolytic pathway. All these
reactions take place in the cytoplasm.

Oxidative phase

The oxidative phase of the pentose phosphate pathway starts with the oxidation of glucose
6-phosphate at carbon 1 with the concomitant formation of NADPH, a reaction catalyzed by glucose 6-phosphate

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 MLS 064: Biochemistry
Module #6 Student Activity Sheet

Name: Class number:

____________________________________________________________ _______
_____ Date:
Section: ____________ Schedule: ________________
________________________________________

dehydrogenase.

This dehydrogenase is highly specific for NADP+. The product of the dehydrogenation is 6-
phosphogluconolactone, which has an ester between the C-1 carboxyl group and the C-5 hydroxyl group. The
next step is the hydrolysis of 6-phosphoglucono-d-lactone by a specific lactonase to give 6-phosphogluconate.
This six-carbon acid is then oxidatively decarboxylated by 6-phosphogluconate dehydrogenase to yield ribulose
5-phosphate. NADP+ is again the electron acceptor. This reaction completes the oxidative phase.

Nonoxidative phase

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 MLS 064: Biochemistry
Module #6 Student Activity Sheet

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____________________________________________________________ _______
_____ Date:
Section: ____________ Schedule: ________________
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The preceding reactions yield two molecules of NADPH and one molecule of ribulose 5-phosphate for each
molecule of glucose 6-phosphat oxidized. The ribulose 5-phosphate is subsequently isomerized to ribose 5-
phosphate by phosphopentose isomerase.

Although ribose 5-phosphate is a precursor to many biomolecules, many cells need NADPH for reductive
biosynthesis much more than they need ribose 5-phosphate for incorporation into nucleotides. For instance,
adipose tissue, the liver, and mammary glands require large amounts of NADPH for fatty acid synthesis. In these
cases, ribose 5-phosphate is converted into glyceraldehyde 3-phosphate and fructose 6-phosphate by
transketolase and transaldolase. These enzymes create a reversible link between the pentose phosphate
pathway and glycolysis by catalyzing these three successive reactions:

The net result of these reactions is the formation of two hexoses and one triose from three pentoses:

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 MLS 064: Biochemistry
Module #6 Student Activity Sheet

Name: Class number:

____________________________________________________________ _______
_____ Date:
Section: ____________ Schedule: ________________
________________________________________

The first of the three reactions linking the pentose phosphate pathway and glycolysis is the formation of
glyceraldehyde 3-phosphate and sedoheptulose 7-phosphate from two pentoses:

The donor of the two-carbon unit in this reaction is xylulose 5-phosphate, an epimer of ribulose 5-phosphate.
Ribulose 5-phosphate is converted into the appropriate epimer for the transketolase reaction by phosphopentose
epimerase:

In the second reaction linking the pentose phosphate pathway and glycolysis, glyceraldehyde 3-phosphate
and sedoheptulose, 7-phosphate react to form fructose 6-phosphate and erythrose 4-phosphate:

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 MLS 064: Biochemistry
Module #6 Student Activity Sheet

Name: Class number:

____________________________________________________________ _______
_____ Date:
Section: ____________ Schedule: ________________
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This synthesis of a four-carbon sugar and a six-carbon sugar is catalyzed by transaldolase. In the third
reaction, transketolase catalyzes the synthesis of fructose 6-phosphate and glyceraldehyde 3-phosphate from
erythrose 4-phosphate and xylulose 5-phosphate:

The sum of these reactions is:

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 MLS 064: Biochemistry
Module #6 Student Activity Sheet

Name: Class number:

____________________________________________________________ _______
_____ Date:
Section: ____________ Schedule: ________________
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Xylulose 5-phosphate can be formed from ribose 5-phosphate by the sequential action of phosphopentose
isomerase and phosphopentose epimerase, and so the net reaction starting from ribose 5-phosphate is

Thus, excess ribose 5-phosphate formed by the pentose phosphate pathway can be completely converted into
glycolytic intermediates. Moreover, any ribose ingested in the diet can be processed into glycolytic intermediates
by this pathway. Evidently, the carbon skeletons of sugars can be extensively rearranged to meet physiologic
needs (see table below).

Regulation of Pentose Phosphate Pathway

1. Key enzyme in the pentose-phosphate pathway is glucose-6-phosphate dehydrogenase.
2. Levels of glucose-6-phosphate dehydrogenase are increased in the liver and adipose tissue when large
amounts of carbohydrates are consumed.
3. Glucose-6-phosphate dehydrogenase is stimulated by NADP+ and inhibited by NADPH and by palmitoyl-
CoA (part of the fatty acid synthesis pathway).

Purpose of Pentose Phosphate Pathway

1. Glutathione helps to prevent oxidative damage to cells by reducing hydrogen peroxide (H O ). (See photo
2 2

below).
2. Pentose phosphate pathway functions as an alternative route for glucose oxidation that does not directly
consume or produce ATP.

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 MLS 064: Biochemistry
Module #6 Student Activity Sheet

Name: Class number:

____________________________________________________________ _______
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Section: ____________ Schedule: ________________
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3. The pentose phosphate pathway produces NADPH for fatty acid synthesis. Under these conditions, the
fructose-6-phosphate and glyceraldehyde-3-phosphate generated in the pathway reenter glycolysis.
4. NADPH is also used to reduce glutathione (γ-glutamylcysteinylglycine).
5. Glutathione is also used to transport amino acids across the membranes of certain cells by the γ-glutamyl
cycle.
6. Generation of ribose-5-phosphate
7. When NADPH levels are low, the oxidative reactions of the pathway can be used to generate ribose-5-
phosphate for nucleotide biosynthesis.
8. When NADPH levels are high, the reversible nonoxidative portion of the pathway can be used to generate
ribose-5-phosphate for nucleotide biosynthesis from fructose-6-phosphate and glyceraldehyde-3-
phosphate.

CHECK FOR UNDERSTANDING

You will answer and rationalize this by yourself. This will be recorded as your quiz. One (1) point will be given to
correct answer and another one (1) point for the correct ratio. Superimpositions or erasures in you answer/ratio
is not allowed. You are given 25 minutes for this activity:

1. In the pentose phosphate pathway, the major products are ____________.

a. Ribulose and NADPH
b. Ribulose and NADH
c. Ribulose and NAD+
d. Ribulose and ATP

2. What is the first reaction of the pentose phosphate pathway?

a. Oxidation of glucose 6-phosphate to 6-phosphoglucono-δ-lactone.
b. Oxidation of 6-phosphogluconate to ketopentose ribulose 5-phosphate.
c. Reduction of 6-phosphoglucono-δ-lactone to glucose 6-phosphate.
d. Reduction of ketopentose ribulose 5-phosphate to 6-phosphogluconate.

3. The pentose-phosphate pathway generates which one of the following?

a. NADH, which may be used for fatty acid synthesis.
b. Ribose 5-phosphate, which may be used for the biosynthesis of ATP.
c. Pyruvate and fructose 1,6-bisphosphate by the direct action of transaldolase and transketolase.
d. Xylulose-5-phosphate by one of the oxidative reactions.

4. Which one out of the following enzymes acts in the pentose phosphate pathway?
a. Aldolase
b. Glycogen phosphorylase
c. Pyruvate kinase

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 MLS 064: Biochemistry
Module #6 Student Activity Sheet

Name: Class number:

____________________________________________________________ _______
_____ Date:
Section: ____________ Schedule: ________________
________________________________________

d. 6-phosphogluconate dehydrogenase

5. Oxidation of 3 molecules of glucose by pentose phosphate pathway results in the production of

____________.
a. 3 molecules of pentose, 4 molecules of NADPH and 3 molecules of CO 2

b. 4 molecules of pentose, 6 molecules of NADPH and 3 molecules of CO 2

c. 3 molecules of pentose, 6 molecules of NADPH and 3 molecules of CO 2

d. 4 molecules of pentose, 3 molecules of NADPH and 3 molecules of CO 2

6. Which one of the following statements is correct about the pentose phosphate pathway?
a. Present in plants but not in animals
b. It generates 6 moles of CO2 for each mole of glucose consumed.
c. It is a reductive pathway that consumes NADH.
d. It generates 38 mol of ATP per mole of glucose consumed.

7. Thiamine pyrophosphate is a coenzyme of what enzyme?

a. Gluconolactonase
b. Glucose-6-phosphate dehydrogenase
c. Transaldolase
d. Transketolase

8. Conversion of xylulose 5-phosphate to ribulose 5-phosphate is catalyzed by ___________.

a. Phosphopentose epimerase
b. Transaldolase
c. Transketolase
d. Phosphopentose isomerase

9. Which of the following statements is correct about the reductive pentose phosphate pathway?
a. It is not reversible.
b. Pentoses can provide glycolytic intermediates.
c. Transaldolase transfers 2 carbon units.
d. Transketolase transfers 3 carbon units.

10. The use of NADPH generated from pentose phosphate pathway cannot be ____________.
a. Used for steroid synthesis.
b. Used for the synthesis of fatty acids.
c. Used for the macrophageal functions.
d. Oxidized in the electron transport chain to provide 38 ATPs.

RATIONALIZATION ACTIVITY (THIS WILL BE DONE DURING THE FACE-TO-FACE INTERACTION)

The instructor will now rationalize the answers to the students. You can now ask questions and debate among
yourselves. Write the correct answer and correct/additional ratio in the space provided.

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 MLS 064: Biochemistry
Module #6 Student Activity Sheet

Name: Class number:

____________________________________________________________ _______
_____ Date:
Section: ____________ Schedule: ________________
________________________________________

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 MLS 064: Biochemistry
Module #6 Student Activity Sheet

Name: Class number:

____________________________________________________________ _______
_____ Date:
Section: ____________ Schedule: ________________
________________________________________

RATIO:_______________________________________________________________________________
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9. ANSWER: ________
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C. LESSON WRAP-UP

AL: Minute Paper

1. What was the most useful or the most meaningful thing you learned in this lesson?
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2. What question/s do you have as we end this session?
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You will now mark (encircle) the session you have finished today in the tracker below. This is simply a visual to
help you track how much work you have accomplished and how much work there is left to do.

You are done with the session! Let’s track your progress.

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