Glycolysis Tutorial
This tutorial is intended to help you
see the chemical reactions that make
up the glycolysis pathway. Read the
lessons and with your mouse click
one (click 1) or two times (click 2)
when instructed.
Biochemistry 411 is a course that sneaks up on the unwary. The lessons ring a familiar tune. You
may think you heard it in your other courses, but metabolism in this course is much more demanding in detail.
To get off to a good start, pick up a pencil and draw the structures. Don’t worry about being geometrically
perfect, a quick sketch is better than no sketch and you wont be graded on artistic talent. Drawing structures lets
you see what is happening. For example, glucose in glycolysis. Draw the straight chain structure of glucose
(click 1). Now, convert glucose into fructose by changing the top two carbons on the molecule (click 1). Note an
–OH groups on C-6 where the first phosphate group attached to glucose (click 1). C-1 in fructose receives a
second phosphate later in the pathway (click 1). The phosphates come from ATP and require kinase enzymes
hexokinase and phosphofructokinase I [PFK-1]. Glucose-6-PO4 is converted into fructose 6-PO4 by an isomerase
enzyme phosphoglucoisomerase [PGI]. See how structures show you what is happening.
O
CHOOH
CH
2 -P-O
H-C-OH
C=O OH
HO-C-H
H-C-OH
H-C-OH O
CH2 OH
-P-O
OH
The second phase of glycolysis is called the triose or payoff phase. In this phase the fructose 1,6-bisphosphate
molecule (click 1) is cut in half (click 1). The top half, i.e., the first 3 carbons with the keto group become
dihydroxyacetone phosphate (DHAP) (click 1). C4-C6, become glyceraldehyde-3-phosphate (click 1). Note that when
the split occurs, the two bonding electron go to the top (click 2) and what was C-4 becomes a carbonium ion with a
positive charge (click 1). The carbonium ion causes a rearrangement of electrons to form a carbonyl group (click 1).
A proton is displaced in the final step (click 1). The glyceraldehyde-3-phosphate goes on to the next step (click 1).
O
CHOOH
CH
2 -P-O
H-C-OH
C=O OH
HO-C-H
H-C-OH
H-C-OH O
CH2 OH
-P-O
OH
O
CH2O-P-O
C=O OH
HO-C-H
H
+
H-C-OH
H-C-OH O
CH2O -P-O
OH
CH2OPO3=
C=O
CH2OH
DHAP
HC=O
H-C-OH
CH2OPO3=
Glyceraldehyde 3-PO4
In the triose or second phase the glyceraldehyde 3-PO4 is converted into pyruvate.
Glyceralaldehyde-3-PO4 is in equilibrium with DHAP via the enzyme triose phosphate isomerase (TPI). This
assures that all of the carbons of glucose will be converted into pyruvate, the final product of the pathway. The
first step in the triose pathway is to oxidize C-1 of glyceraldehyde-3-PO4. The glyceraldehyde-3-PO4 then
becomes 3-phosphoglyceric acid (3PGA) (click 2). The slanted arrows show C-1 before and after oxidation.
There is concern, however. The cell must somehow devise a way to conserve the oxidation energy. Click 1 to
see how the energy is conserved. Note that to preserve free energy the cell adds phosphate to C-1
simultaneously with the oxidation by NAD+ (click 1). This creates a high energy phosphate bond (click 1) in
the intermediate 1,3-bisphosphoglycerate (1,3-BPG) that is used to synthesize ATP from ADP (click 1). The
enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the first reaction (click 1) and
phosphoglycerate kinase(PGK) catalyzes the second (click 1). Both of these reactions are freely reversible.
Click 1 to go on.
CHO
PO4
H-C-OH
O
C ~OPO3
H-C-OH
H-C-OH
+
ADP
NAD
CH
OPO
CH2OPO3
2
3
NADH
+ H+
Glyceraldehyde-3-PO4
dehydrogenase
COO
ATP CH2OPO3
Phosphoglycerate
kinase
To end the triose phase, 3-phosphoglycerate is converted into pyruvate (click 1). By now you
should be able to predict that the phosphate on C-3 of 3PGA will used to make ATP. This is not obvious at
first because the phosphate ester in 3PGA is not high energy (click 1). Therefore, to make ATP this phosphate
must be converted into one that is high energy. That high energy molecule is phosphoenolpyruvate (PEP)
(click 1). Your chore now is figure how to make PEP from 3PGA. A clue comes when you see that the
phosphate in PEP in on C-2. Therefore, you must first shift the phosphate on 3PGA to make 2PGA (click 1).
This is done by a mutase enzyme, phosphoglycerate mutase (PGM). To make the phosphate in 2PGA high
energy, a H2O must be removed from 2PGA to form an enol-phosphate bond (click 1). Enol-phosphate is a
high energy phosphate, in fact one of the highest in terms of release of free energy (click 1). From PEP it is
simply a matter to transfer the phosphate to ADP (click 1),which brings us to the end of the pathway with the
formation of ATP and pyruvate.
COO
H-C-OH
Low energy
CH2OPO3
3-PGA
COO
H-C-OPO3 -H2O
COO
High energy
C~OPO3
CH2OH
CH2 ADP
2-PGA
PEP
COO
C=O
ATP CH3
Pyruvate
What did you learn? Answer the following questions regarding glycolysis and energy.
1. What is the overall energy yield in ATP production when one molecule of glucose is
converted to pyruvate? (click for answer).
There is a net of 2 ATPs synthesized for each glucose molecule. Two ATPs are required to
bring the glucose to the fructose 1,6-bisphosphate stage (-2). One ATP each is synthesized
when glyceraldehyde-3-PO4 goes to 3PGA and PEP to pyruvate (+2). Since the latter reaction
occurs with each triose, the total is +4 for all the carbons of glucose. -2 + (+4) = +2
2. Based on free energy yield, predict the reactions that are irreversible or poorly reversible in
the glycolysis pathway. (click for answer)
There are 3. Glucose to glucose 6-PO4 catalyzed by hexokinase; fructose 6-PO4 to
fructose 1,6 bisPO4 catalyzed by phosphofructokinase-I, and PEP to pyruvate catalyzed
by pyruvate kinase. All 3 are catalyzed by kinase enzymes and all three have very large
negative delta G values (check your textbook for delta G values).
3. Why is the phosphoglycerate kinase reaction reversible? What about the other kinase reactions?
(click for answer)
The product, glycerate 1,3-bisPO4 is high energy. Thus, it can yield ATP by transferring
the phosphate group to ADP. The other kinase reactions all yield low energy phosphate esters
as products and hence cannot be reversed. Reversing would mean forming a high energy ATP
phosphoanhydride bond in ATP from a low energy phosphate ester compound and this is not
energetically feasible.