Topic : GLYCOLYSIS
Lecturer: Dra. Uy
GLYCOLYSIS
-breakdown of GLUCOSE to 2 moles of PYRUVATE
(and LACTATE in RBCs remember, in the absence of
oxygen)
-occurs in the CYTOSOL of ALL CELLS
-yields a total of 40 ATP under AEROBIC CONDITIONS
(uses 2 in the process so NET ATP is 38)
-yields 2 ATP under ANAEROBIC CONDITIONS
-requires Magnesium ion as cofactor
Here’s the big picture of what Glycolysis, aerobic and
anaerobic conditions are and why these are important:
After eating rice or any other food which is essentially
CARBOHYDRATES, it is converted to GLUCOSE.
Glucose then undergoes GLYCOLYSIS. The important
products of this glycolysis are :
ATP, NADH and PYRUVATE.
Let’s analyze the products of Glycolysis.
(1)ATP: ATP is already a final product, meaning it’s
already functional and will be used by the cell for
energy-requiring activities. Notice that we have not yet
involved the electron transport chain to generate ATP
and this is what is meant by the term substrate-level
phosphorylation.
(2)NADH: NADH, in the presence of oxygen (aerobic
condition) will be used in the ELECTRON TRANSPORT
CHAIN (ETC) to generate ATP. Each mole of NADH is
equivalent to 3 ATP. Since we already enter the ETC,
this is now called oxidative phosphorylation.
(3) PYRUVATE, by the action of pyruvate
dehydrogenase complex will be converted to acetyl-
coA. Acetyl-coA will then enter the carboxylic acid
cycle/Krebs cycle. The Krebs cycle will generate 12
moles of ATP per cycle and will involve oxidative
phosphorylation.
Notice that in the absence of Oxygen, NADH and
Pyruvate cannot be utilized as products of glycolysis and
we can only produce 2 ATP via the substrate level
phosphorylation. This is known as anaerobic
respiration and occurs in states like heavy exercise
where oxygen is already depleted because of too much
cellular activity.
Now that we have a general picture of what glycolysis
is, we will be discussing the glycolytic pathway step by
step.
REVIEW on Carbohydrate structure
To fully understand and appreciate the enzymes, the structures and
the biochemical processes, it might be necessary to discuss some
structure and concepts about carbohydrates and glucose.
Carbohydrates are simply Carbon (C) plus
Water(hydrate, H2O)
C + H2O = C-H2O
So if we have a carbohydrate compound, we sort of like
have a compound with lots of Carbon and Water joined
together.
C-H2O- C-H2O C-H2O- C-H2O C-H2O- C-H2O C-H2O- C-H2O
C-H2O- C-H2O C-H2O- C-H2O C-H2O- C-H2O C-H2O- C-H2O
Glucose is a simple carbohydrate. That means glucose
must also be composed of Carbon and Water as well.
Glucose has six carbons. Six carbons means we also
have six water molecules. So,
1 C-H
2O
I
2 C-H2O
3 I
C-H2O
4 I
C-H2O
5 I
C-H2O
6 I
C-H2O
Also, glucose, as we learned previously, is a
carbohydrate sugar with an Aldehyde and an Alcohol
group. So to complete the structure of glucose, we need
to show these groups.
Let’s take care of the alcohol group first.
Alcohol is a compound with a functional group
represented as R-OH.
R here can be substituted with any organic compounds
like CH3-, CH2CH3-. For example, in R-OH, if we replace R
with CH3, (methyl),we get
CH3-OH or what we commonly known as methyl
alcohol. If we replace R with CH3CH2 (ethyl) we get
CH3CH2-OH or ethyl alcohol, the alcohol you use in your
hand sanitizers and rubbing alcohol.
In our illustration in the previous page, notice that if we
place an additional Hydrogen in carbon number 6, we
will arrive at a structure similar to this :
1 C-H
2O
I
2 C-H2O
3 I
C-H2O
4 I
C-H2O
5 I
C-H2O additional H
6 I
C-H2O-H
It follows the alcohol pattern of R-OH right?
What about the aldehyde group? Aldehydes are
compounds which follows the pattern R-CHO.
Again, R can be any compound. For example if we
replace R with CH3 we get CH3-CHO or methyl aldehyde.
If we replace R with Hydrogen, we get H-CHO or what
we commonly know as formaldehyde, or the formalin
we use in the cad room. 
So what happens if we take one Hydrogen from the first
carbon in our illustration above? We get a structure
similar to this :
remove H
1 C-HO
I
2 C-H2O
3 I
C-H2O
4 I R group
C-H2O
5 I
C-H2O
6 I
C-H2O-H
If you consider the boxed structure as the R group, we
now follow the aldehyde pattern R-CHO. Notice that
the Hydrogen we removed from the first carbon to
make the aldehyde group is the same hydrogen we add
in the sixth carbon to make the alcohol group. So we
have the carbon, the hydrate (water), the aldehyde, the
alcohol and so we have the final glucose structure.
Now what if a certain HEXOKINASE enzyme acts on our
glucose?
If we breakdown the name of the enzyme, we will have
a clue as to what it does. HEXO means 6, so probably,
this enzyme acts on compounds with 6 carbons.
KINASE, remember, always transfers a PHOSPHATE
group usually from ATP to the compound it is acting on.
So if a HEXOKINASE acts on our glucose what will
happen? Hexokinase will transfer one phosphate group
from ATP to our glucose. Since ATP loses a phosphate
group it now becomes a diphosphate or ADP. And our
glucose after receiving the phosphate from ATP will
become glucose-phosphate. And since phosphate will
be attached on the sixth carbon, it is specifically called
glucose-6-phosphate.
1 C-HO
I
2 C-H2O
3 I
C-H2O
4 I
C-H2O
5 I PO4 on Carbon 6
C-H2O
6 I
C-H2O-PO4
If an additional phosphate group is added just beside
the phosphate group already attached, then our
compound is called glucose-6-diphosphate. But if an
additional phosphate is attached in another carbon, say
Carbon number 1, then our compound will be called,
glucose-1,6-bisphosphate.
Glycolysis : the steps.
(1)So the carbohydrate from the rice we ate was
digested to glucose, glucose in turn will be acted upon
by our first enzyme HEXOKINASE. From our review
above, we know that hexokinase will convert glucose to
glucose-6-phosphate (G6P).
HEXOKINASE
GLUCOSE GLUCOSE-6-Phosphate
ATP  ADP
What you need to know about this step:
-Hexokinase has a low Km (very high affinity for
glucose) and will be inhibited by high concentrations of
Glucose-6-phosphate as a regulatory mechanism. When
this happens, another enzyme, glucokinase (GCK), not
inhibited by G6P, will act on excess glucose to prevent
hyperglycemia.
-remember that glucokinase activity REQUIRES
ENERGY in preparation for conversion to glycogen, the
storage form of glucose. You store only when there is
abundant supply. So during the starved state, you do
not store, because there is nothing to store in the first
place, and the activity of glucokinase decreases.
-genetic defect in GLUCOKINASE is present in a disease
called MODY (Maturity-Onset Diabetes of the Young)
Type 2 whose primary symptom is hyperglycemia
(2) The next step is the conversion of glucose-6-
phosphate to fructose-6-posphate via the enzyme
phosphoglucose isomerase
Phosphoglucose isomerase
GLUCOSE-6-phosphate fructose-6-Phosphate
(3) Fructose-6-phosphate will then be Phosphorylated
again. Since this is phosphorylation we have a clue that
the enzyme involved is a KINASE. This KINASE will act on
a substrate which is fructose that has a phosphate
attached to its 6th carbon. The name of the enzyme
therefore is PHOSPHOFRUCTOKINASE 1 (PFK1). This
enzyme will add phosphate to the first carbon of
fructose-6-phosphate so we would end up with a
compound named fructose-1,6-bisphosphate.
PHOSPHOFRUCTOKINASE
FRUCTOSE-6-phosphate FRUCTOSE-1,6-bisphosphate
ATP  ADP
What you need to know about this step:
- this is an IRREVERSIBLE and the REGULATORY STEP of
glycolysis
- deficiency in phosphofructokinase leads to
accumulation of glycogen and manifests as HEMOLYTIC
anemia an muscle cramping
Notice that in steps 1 and 3 we’ve used a total of 2
ATPs. This is known as the preliminary or the
investment stage of glycolysis. This means that we are
investing ATP to produce more ATPs.
(4) The next step is known as the splitting stage.
Splitting means we will split our 6-carbon sugar into two
3-carbon sugar (triose). Fructose-1,6-bisphosphate will
be split to 2 triose sugars namely D-glyceraldehyde-3-
phosphate (GADP) and dihydroxyacetone phosphate
(DHAP)by the enzyme ALDOLASE. These two sugars are
ISOMERS.
ALDOLASE
FRUCTOSE-1,6-bisphosphate GADP + DHAP
(5) DHAP can be converted to GADP by the enzyme
Triose Phosphate Isomerase. So in essence, we produce
2 GADPs.
What you need to know about this step:
-Aldolase A is found in muscles and RBCs and deficiency
of which causes nonspherocytic hemolytic anemia
-deficiency in Triose phosphate isomerase leads to
neonatal hemolytic anemia and progressive neurologic
inolvement
After the splitting stage, we are now ready to collect
the ATPs. 
(6) Each GADPs we produce will undergo
dehydrogenation and addition of inorganic phosphate
using the enzyme glyceraldehyde-3-phosphate
dehydrogenase (GADPH). This step yields 1,3-
bisphosphoglycerate (1,3-BPG) and NADH.
GADPH
GADP 1,3-BPG + NADH
*GADPH is inhibited by SULFHYDRYL AGENTS
(7) Notice that on our previous product, 1,3-BPG*, we
have two phosphate groups. In the next reaction we will
transfer one of these phosphate group to ADP which
means we will generate ATP from ADP via the enzyme
Phosphoglycerate Kinase(PGK). Because 1,3-BPG loses
a phosphate group it now called 3-phosphoglycerate
(3-PG)
PGK
1,3-BPG 3-PG
ADP  ATP
*1,3-BPG can also be converted to 2,3-BPG via the a
MUTASE enzyme. 2,3-BPG binds greatly in
deoxygenated Hgb. It can be converted to 3-PG to
enter the glycolytic pathway again via the enzyme
Phosphatase.
(8) 3-PG will undergo isomerization and will be
converted to 2-Phosphoglycerate (2-PG)
(9) 2-PG under the action of Enolase will be converted
to Phosphoenol Pyruvate (PEP).
*ENOLASE is INHIBITED by FLUORIDE.
(10) Finally, PEP by the action of the enzyme pyruvate
kinase will be dephosphorylated and its phosphate
group will be donated to ADP to form ATP. This is our
second substrate level phosphorylation.
Pyruvate kinase
PEP Pyruvate
ADP  ATP
Deficiency in Pyruvate Kinase leads to poor production
of ATP (ADP has higher concentrations than ATP) and
consequently affecting the ion pumps (esp Na-K
ATPase) which causes the cells to swell and lyse leading
to hemolytic anemia.
ATP COUNT Regulation of Glycolysis:

For one glucose molecule : -Under the FED STATE, there is HIGH LEVEL of INSULIN
and LOW LEVEL OF GLUCAGON. This causes decreased
ATP Loss : cAMP and consequently decreased level of protein
In step 1, we used 1 ATP. kinase A. This decreased level of protein kinase A
In step 3, we used another ATP. causes dephosphorylation or activation of PFK2 in the
Total ATP used : 2 bifunctional enzyme PFK2/FBP2. Activation of PFK2
causes formation of Fructose-2,6-bisphosphate.
ATP Gained : Elevated levels of Fructose-2,6-bisphosphate leads to
In Step 6, we generated 1 NADH. Remember that increased glycolysis because it is a POTENT ACTIVATOR
Glucose was split into two triose? of Phosphofructokinase 1.
So that means 1NADH x 2 = 2NADH
Total NADH generated : 2 This NADH can enter the Bottomline : Fed state, insulin and Fructose-2,6-
Krebs cycle under aerobic conditions. bisphosphate favors GLYCOLYSIS.
Each NADH is equal to 3ATP.
So 2 NADH x 3 = 6 Inhibitors of Phosphofructokinase :
Total ATP generated = 6 - Citrate
- ATP
In Step 7, we generated 1 ATP. (x2 since we split - Hydrogen ions (low pH/acidic environment)
glucose into two)
In Step 10, we generated 1 ATP. (x2 since we split Activators of Phosphofructokinase:
glucose into two) - AMP
Total ATP Generated = 4 - Fructose 2,6-bisphosphate

Our final product is pyruvate (x2). Pyruvate will enter

the Krebs cycle. But before that, it should be converted
to acetyl-coA first by pyruvate dehydrogenase
complex. This step produces 1 NADH (x2).
Each NADH is equal to 3ATP.
So 2NADH x 3 =6
Total ATP Generated : 6

When the 2 pyruvates we have had been converted to

acetyl-coA, each oh these two acetyl-coA will generate
12 ATP after entering the Krebs Cycle. So 12 X 2 =24
Total ATP generated : 24

TOTAL ATP Generated for 1 glucose molecule : 40

TOTAL ATP used : 2
NET ATP Generated : 38

Under anaerobic conditions, we do not have oxygen

which means we cannot proceed with the Krebs cycle
and the only way we can generate ATP is via steps 7 and
10. That means we generate 4 ATPs but since we use 2
ATPs in steps 1 and 3, our NET ATP gain for anaerobic
condition is only 2.