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Last edited: 8/8/2021

GLYCOLYSIS
Glycolysis: Steps and Energy Yield Medical Editor: Jona Frondoso

OUTLINE (ii) Energy Generation Phase (Steps 6 to 10)

I) INTRODUCTION • Net of two molecules of ATP are formed by
substrate-level phosphorylation per glucose
II) ENERGY INVESTMENT PHASE metabolized
III) ENERGY GENERATION PHASE o Substrate-level phosphorylation – Energy
needed for the production a high-energy
IV) ENERGY YIELD FROM GLYCOLYSIS phosphate (e.g. phosphate in ATP) comes
V) APPENDIX from a substrate rather than from the electron
transport chain
VI) REVIEW QUESTIONS
VII) REFERENCES
II) ENERGY INVESTMENT PHASE

(A) STEP 1: PHOSPHORYLATION OF GLUCOSE

I) INTRODUCTION
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(1) Glucose
● Monosaccharide, 6-carbon molecule
● End-product of digestive breakdown of carbohydrates
● Water-soluble solute, hence can be transported through
the cell membrane via passive diffusion through the
bidirectional GLUT transporters
o Different GLUT Transporters
All are insulin-independent except GLUT IV which is
insulin-dependent (Figure 1)
• GLUT I: RBCs, fetus, blood brain barrier
• GLUT II: kidney, liver, pancreas
• GLUT III: placenta, neurons, kidney
• GLUT IV: muscle, adipose

Remember:

Figure 2. Energy investment phase: phosphorylation of

glucose.

● There are no specific transmembrane carriers for

phosphorylated glucose, hence they do not readily
penetrate the cell membrane.
● Irreversible phosphorylation of glucose effectively trapping
the sugar as cytosolic glucose 6-phosphate (G6P) (Figure
2)
● Catalyzed by several isozymes of hexokinase
● Mammals have 4 isozymes of hexokinase (Table 1)
o Hexokinase I, II, III
o Hexokinase IV (aka Glucokinase)

Figure 1. Different GLUT transporters. Table 1. Hexokinase Versus Glucokinase [Harvey & Ferrier, 2011]
Hexokinase Glucokinase
(2) Glycolysis Distribution Most tissues Liver parenchymal
cells and β cells of
• Employed by all tissues in which glucose is broken down
the pancreas
to provide energy (in the form of ATP) and intermediates
for other metabolic pathways Km Low (and, High 9and,
therefore, a high therefore, a low
• End-product of oxidation of glucose in aerobic cells is
affinity for glucose) affinity to glucose)
pyruvate (3-carbon molecule)
• Occurs in the cytoplasm Vmax Low High
• First metabolic pathway in the cytosol Substrate Several hexoses in Glucose only
• Sequence of 10 steps subdivided into two stages [Harvey &
addition to glucose
Ferrier, 2011] Inhibition Yes No
by G6P
(i) Energy Investment Phase (Steps 1 to 5) Induced by No Yes
• Phosphorylated forms of intermediates are Insulin
synthesized at the expense of ATP
o ATP → ADP + Pi
o Pi or inorganic phosphate is transferred to a
substrate to be phosphorylated

GLYCOLYSIS METABOLISM: Note #1. 1 of 4

 (B) STEP 2: ISOMERIZATION OF GLUCOSE 6- III) ENERGY GENERATION PHASE
PHOSPHATE
(A) STEP 6: OXIDATION OF GLYCERALDEHYDE 3-
PHOSPHATE

Figure 3. Aldose-ketose isomerization of glucose 6-phosphate

to fructose 6-phosphate.
● Isomerization of glucose 6-phosphate (G6P) to fructose 6-
phosphate (F6P) (Figure 3)
Figure 5. Energy generation phase: Oxidation of
o G6P – contains an aldehyde group Glyceraldehyde 3-phosphate to 1,3-Bisphoshphoglycerate.
o F6P – contains a ketone group AfraTafreeh.com
● Catalyzed by phosphohexose isomerase
● Readily reversible and not a rate-limiting or regulated step ● Conversion of glyceraldehyde 3-phosphate to 1,3-
bisphosphoglycerate (1,3-BPG) by glyceraldehyde 3-
phosphate dehydrogenase (Figure 5)
(C) STEP 3: PHOSPHORYLATION OF FRUCTOSE 6- o Coupled with the attachment of an inorganic phosphate
PHOSPHATE (Pi), a high-energy phosphate, at carbon 1 of G3P
o NAD+ rips off hydrides from G3P producing NADH

Remember:
● Hydride has two electrons instead of just 1 electron.

Figure 6. Atomic structure of hydride.

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Figure 4. Energy investment phase: Conversion of fructose 6- ● First oxidation-reaction of glycolysis
phosphate to triose phosphates. • The amount of NAD+ in the cell is limited; therefore, the
NADH from this reaction must be re-oxidized to NAD+ for
● Most important control point and the rate-limiting step glycolysis to continue [Harvey & Ferrier, 2011]
and committed step of glycolysis ● Major mechanisms of oxidizing NADH are
● Irreversible phosphorylation of carbon 1 producing fructose o NADH-linked conversion of pyruvate to lactate
1,6-bisphosphate (F1,6-BP) (Figure 4) (anaerobic)
o Bisphosphate means there are carbon spaces o Oxidation of NADH via the electron transport chain
between the two phosphates, while biphosphate (aerobic)
means they are next to each other.
● Catalyzed by phosphofructokinase-1 (PFK)
(B) STEP 7: SYNTHESIS OF 3-PHOSPHOGLYCERATE
PRODUCING ATP
(D) STEP 4: CLEAVAGE OF FRUCTOSE 1,6-
BISPHOSPHATE
• Aldolase cleaves fructose 1,6-bisphosphate (F1,6-BP) to
dihydroxyacetone phosphate (DHAP) and glyceraldehyde
3-phosphate (G3P) (see Figure 4)
• Reaction is reversible and not regulated

(E) STEP 5: ISOMERIZATION OF Figure 7. Energy generation phase: Conversion of 1,3-

DIHYDROXYACETONE PHOSPHATE Bisphoshphoglycerate to 3-phosphoglycerate.
• Triose phosphate isomerase interconverts
dihydroxyacetone phosphate and glyceraldehyde 3-
phosphate (see Figure 4) ● Conversion of 1,3-BPG to 3-phosphoglycerate (3-PG)
• DHAP is isomerized to G3P since the latter is more stable (Figure 7)
and is required for the pathway to proceed ● Catalyzed by phosphoglycerate kinase
• Results in the net production of two molecules of G3P o Physiologically reversible, unlike most kinases
● High-energy phosphate group of 1,3-BPG is used to
FYI: synthesize 2ATP from 2ADP
● At this point of the glycolytic pathway, we are now dealing o Replaces the two ATP molecules consumed by the
with two molecules of glyceraldehyde 3-phosphate (G3P). earlier formation of G6P and F1,6-BP
● Therefore, each succeeding step will result to two o Example of substrate-level phosphorylation
molecules of the intermediate or product

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(C) STEP 8: SHIFT OF THE PHOSPHATE GROUP o Lactic acid causes the blood pH to become
FROM CARBON 3 TO CARBON 2 more acidic (↓pH), and may lead to
metabolic acidosis

Figure 8. Energy generation phase: Conversion of 3-

Bisphoshphoglycerate to 2-phosphoglycerate.

• Shift of phosphate group from carbon 3 to carbon 2 of

phosphoglycerate by phosphoglycerate mutase (Figure
8)
• Freely reversible step
(D) STEP 9: DEHYDRATION OF 2-
PHOSPHOGLYCERATE Figure 11. Pyruvate pathways in aerobic and anaerobic
conditions.

Nice to Know:
● ↑ LDH levels can be seen in certain conditions

Figure 9. Energy generation phase: Conversion of 2- Figure 12. Conditions where LDH can be seen.
phosphoglycerate to phosphoenolpyruvate.

● Dehydration of 2-phosphoglycerate (2-PG) by enolase IV) ENERGY YIELD FROM GLYCOLYSIS

redistributes the energy within the 2-phosphoglycerate
molecule (Figure 9) (A) NET PRODUCTS OF AEROBIC GLYCOLYSIS
● Results in the formation of the high-energy compound, o Glucose → 2 Pyruvate
phosphoenolpyruvate (PEP) o 2 ADP → 2 ATP
● Reversible step o 2 NAD+ → 2 NADH
(E) STEP 10: FORMATION OF PYRUVATE PRODUING (B) ATP YIELD
ATP
(1) ATP from Substrate-Level Phosphorylation

(i) Energy Investment Phase = -2 ATP

• -1 ATP from Step 1 (Phosphorylation of glucose)
• -1 ATP from Step 3 (Phosphorylation F6P)

(ii) Energy Generation Phase = +4 ATP

• +2 ATP from Step 7 (Synthesis of 3-PG)
• +2 ATP from Step 10 (Formation of pyruvate)
Figure 10. Energy generation phase: Conversion of (2) ATP from Oxidative Phosphorylation
phosphoenolpyruvate to pyruvate. ● Oxidative Phosphorylation – ATP generation as a result
● Conversion of PEP to pyruvate catalyzed by pyruvate from the transfer of electrons from NADH and FADH2 to
kinase (Figure 10) oxygen by a series of electron carriers
o Equilibrium of the reaction favors ATP formation ● NADH obtained from Step 6 (Oxidation of G3P)
o Another example of substrate-level phosphorylation • Reducing equivalents from 2 NADH are transferred to

● Third irreversible reaction of glycolysis electron transport chain in two ways [Harvey & Ferrier, 2011]
● Pyruvate can divert into two different pathways (Figure
(i) Glycerophosphate Shuttle
11)
• 4 ATP will be formed
(1) Aerobic Conditions • 2 ATP from glycolysis + 4 ATP from ETC = 6 ATP
• Occurs in cells adequate oxygen supply
• Conversion of pyruvate to Acetyl-CoA, a major fuel in TCA (ii) Malate-Aspartate Shuttle
(or citric acid) cycle • 6 ATP will be formed
(2) Anaerobic Conditions • 2 ATP from glycolysis + 6 ATP from ETC = 8 ATP

• Occurs when there is scarcity in oxygen supply

• Conversion of pyruvate to lactate catalyzed by lactate
dehydrogenase
o NADH unload their hydrides onto specific molecules to
take it to the electron transport chain.
o During anaerobic conditions, NADH unloads their
hydrides onto pyruvates.
o Pyruvate gets reduced to lactic acid
o Lactic acid can go to the liver and be
converted to either glucose or ATP

GLYCOLYSIS METABOLISM: Note #1. 3 of 4

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V) APPENDIX VI) REVIEW QUESTIONS

Which one of the following statements concerning
glycolysis is NOT TRUE?
a. It occurs in the cytoplasm.
b. GLUT transporters found in the adipose tissue are
insulin-dependent.
c. The conversion of glucose to lactate does not require
oxygen.
d. The conversion of glucose to lactate yields two ATP
and two NADH.
e. Glycolysis in the liver is catalyzed by glucokinase.

The reaction catalyzed by phosphofructokinase-1

a. is the committed step of the glycolytic pathway
b. produces fructose 1,6-biphosphate
c. uses ATP producing AMP
d. is part of the generation phase of the glycolytic
pathway
e. uses fructose 1-phosphate as substrate
Which of the following enzymes catalyzes the
conversion of pyruvate to lactate?
a. Lactate carboxylase
b. Lactate dehydrogenase
c. Pyruvate dehydrogenase
d. Lactate kinase
e. Pyruvate isomerase

Which of the following statements concerning the

generation phase of glycolysis is correct?
a. 4 NADH are produced from the oxidation of
glyceraldehyde 3-phosphate.
b. Substrate-level phosphorylation occurs only once
during the generation phase.
c. The end-product are two pyruvates.
d. Dihydroxyacetone phosphate is the substrate of the
generation phase.
e. It is also known as the energy investment phase.

What is the total ATP yield from aerobic glycolysis if

the reducing equivalents from NADH are transferred to
Figure 13. 10 steps of glycolysis. [Glycolysis, 2011]
the electron transport chain via the glycerophosphate
shuttle?
a. 2 ATP
b. 3 ATP
c. 4 ATP
d. 5 ATP
e. 6 ATP

VII) REFERENCES
● Glycolysis. (2021, August 2). Retrieved from Pinterst:
https://www.pinterest.ph/pin/342906959131813599/
● Harvey, R., & Ferrier, D. (2011). Lippincott's Illustrated Reviews
- Biochemistry (5th ed.). Philadelphia: Lippincott Williams & WIlkins.

Figure 14. Important facts about glycolysis.

4 of 4 METABOLISM: Note #1. GLYCOLYSIS

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