Last edited: 11/14/2021

REGULATION OF GLYCOLYSIS
Regulation of Glycolysis: Hexokinase, PFK-1, & Pyruvate Kinase Medical Editor: Jona Frondoso

OUTLINE (B) TYPES OF REGULATION

I) INTRODUCTION
II) REGULATION OF HEXOKINASE
III) REGULATION OF PHOSPHOFRUCTOKINASE-1
IV) REGULATION OF PYRUVATE KINASE
V) APPENDIX
VI) REVIEW QUESTIONS
I) INTRODUCTION
(A) ENZYMES OF THE GLYCOLYTIC PATHWAY
(1) Highly Regulated Glycolytic Enzymes
There are three highly regulated enzymes in the glycolytic
pathway.
Catalyzes the irreversible steps in glycolysis
(i) Hexokinase
o Catalyzes the irreversible conversion of glucose to
glucose 6-phosphate (G6P)
(ii) Phosphofructokinase-1
o Catalyzes the irreversible phosphorylation of fructose
6-phosphate (F6P) to fructose 1,6-phosphate (F1,6-
P)
(iii) Pyruvate Kinase
o Catalyzes the irreversible conversion of
phosphoenolpyruvate (PEP) to pyruvate
(2) ATP Generating Enzymes of Glycolysis
Net ATP yield is 2 ATP per glucose
(i) Phosphoglycerate Kinase
o Catalyzes the conversion of 1,3-bisphosphoglycerate
(1,3-BPG) to 3-phosphoglycerate (3-PG)
o Yields 2 ATP per glucose
(1) Allosteric Regulation
Enzyme has an overall three-dimensional shape with an
active site and an allosteric site.
Enzyme can either be allosterically activated or inhibited.
(i) Active site
o Binds to the actual substrate
(ii) Allosteric site
o Site other than the active site which can control the
overall three-dimensional shape of the enzyme
resulting to the activation or inhibition of the enzyme
(2) Hormonal Regulation
Covalent modification of regulatory enzymes through
phosphorylation / dephosphorylation
Seen in insulin and glucagon
II) REGULATION OF HEXOKINASE
(A) ALLOSTERIC REGULATION
(1) Hexokinase
Activator: ↑ glucose
Inhibitor: ↑ glucose 6-phosphate
(2) Glucokinase
Activator: ↑ glucose
o Brings glucokinase out of the liver allowing it to
stimulate the phosphorylation of glucose to G6P
Inhibitor: ↑ fructose 6-phosphate
o Puts glucokinase back into the nucleus (Figure 1)

(ii) Pyruvate Kinase

o Catalyzes the irreversible conversion of PEP to
pyruvate
Figure 1. Allosteric regulation of hexokinase and glucokinase.
o Yields 2 ATP per glucose
(3) NADH generating enzymes of glycolysis Remember:

Net NADH generated is 2 NADH per glucose Hexokinase

o Found in most tissues
(i) Glyceraldehyde 3-phosphate dehydrogenase o Freely circulating in the cytoplasm
o Hexokinase II – predominant in the cytoplasm
o Catalyzes the conversion of glyceraldehyde 3-
phosphate dehydrogenase Glucokinase
o Found only in the liver until brought out by a
o Yields 2 NADH per glucose
stimulus
(4) ATP Investing Enzymes of Glycolysis o Housed in the nucleus
Total of ATP used is 2 ATP per glucose

(i) Hexokinase
o Catalyzes the irreversible conversion of glucose to
G6P
o Uses 1 ATP per glucose

(ii) Phosphofructokinase-1
Catalyzes the irreversible phosphorylation of F6P to
F1,6-P
Uses 1 ATP per glucose

REGULATION OF GLYCOLYSIS METABOLISM: Note #2 1 of 4

 (B) HORMONAL REGULATION (B) REGULATION OF FRUCTOSE 2,6-
BISPHOSPHATE LEVELS
(1) Insulin
Fructose 2,6-bisphosphate – most powerful regulator of
Released from pancreatic beta cells when glucose levels PFK-1
are high
Activates: hexokinase and glucokinase (synthesis
increases) (1) Phosphofructokinase-2 (PFK-2) / Fructose 2,6-
Stimulates glycolysis, inhibits gluconeogenesis bisphosphatase (F2,6-BPase) Control of Glycolysis

(2) Glucagon Heteronuclear or binuclear enzyme – an enzyme complex

Released from the pancreatic alpha cells when glucose
levels are low
Inhibits: hexokinase and glucokinase
Stimulates glycolysis, inhibits gluconeogenesis (Figure 2)
with two different types of domains
Can be allosterically and hormonally regulated (Figure 4)
(i) Allosteric Regulation
o Small amount of fructose 6-phosphate (F6P) can
divert from the glycolytic pathway and reacts to PFK-
2 / F2,6-BPase complex
o Binds to PFK-2 domain forming F2,6-BP
o Binds to F2,6-BPase domain forming F6P

(ii) Hormonal Regulation

Figure 2. Hormonal regulation of hexokinase and glucokinase.
FYI:
Insulin – released when blood glucose level >130mg/dL
Glucagon - released when blood glucose <70mg/dL
III) REGULATION OF PHOSPHOFRUCTOKINASE-1
(A) ALLOSTERIC REGULATION
(1) Allosteric Inhibitor
(i) Adenosine triphosphate (ATP)
o ↑ ATP = ↑ energy supply
o Since cells can only store so much ATP, ATP
generated from the electron transport chain can
allosterically bind to PFK-1 inhibiting it.
(ii) Citrate
o Krebs Cycle intermediate
o ↑ citrate = ↑ Krebs cycle activity = ↑ NADH, FADH2
= ↑ ATP = ↑energy supply
o Well-fed state
 Favors glycolysis
 ↑ Insulin, ↓ glucagon
 Activates PFK-2 domain, inhibits F2,6-BPase
domain by dephosphorylation of the enzyme = ↑
F2,6-BP = activates PFK-1 = ↑ conversion of F6P
to F1,6-BP
• Insulin dephosphorylates PFK-2/F2,6BP by
activating phosphoprotein phosphatase
(Table 1)
o Fasting state
 Does not favor glycolysis
 ↑ Glucagon, ↓ insulin
 Activates F2,6-BPase domain, inhibits PFK-2
domain by phosphorylation of the enzyme = ↓
F2,6-BP = ↓ activation PFK-1 = ↓ conversion of
F6P to F1,6-BP
• Glucagon phosphorylates PFK-2/F2,6BP by
activating cAMP-dependent kinases (Table 1)

(iii) Fructose 2,6-bisphosphate (F2,6-BP)

o Activates PFK-1 leading to conversion of more
fructose 6-phosphate to fructose 1,6-bisphosphate to
make more ATP
Remember: Figure 4. Hormonal regulation of phosphofructokinase-1.

Fructose 2,6-bisphosphate Remember:

o Has a role in gluconeogenesis
o When its concentration decreases, it also
stimulates fructose 1,6-bisphosphatase which Table 1. Hormonal Regulation of PFK-2 and F1,6-BPase
eventually converts that to glucose. Enzyme Phosphorylated Dephosphorylated
Domain by glucagon by Insulin
(2) Allosteric Activator PFK-2 inactive active

(i) Adenosine diphosphate (ADP) F2,6-BPase active inactive

o ATP is broken down into ADP and inorganic

phosphate by hydrolysis
o ↑ ADP = ↓ ATP = ↓ energy supply

Figure 3. Allosteric regulation of phosphofructokinase-1.

2 of 4 METABOLISM: Note #2 REGULATION OF GLYCOLYSIS

 IV) REGULATION OF PYRUVATE KINASE
Nice to Know:
(A) ALLOSTERIC REGULATION
(1) Allosteric Activator
(i) Fructose 1,6-bisphosphate (F1,6-BP)
o An example of feed forward reaction
 Feed forward reaction – Metabolite produced
early in the pathway activates an enzyme that
catalyze a reaction further down the pathway
o Signals the pyruvate kinase to get ready and increase
its activity since its concentration increased (Figure 5)
Arsenate Poisoning
o Arsenate can prevent net ATP and NADH production by
glycolysis, without inhibiting the pathway itself
o Competes with organic phosphate as a substrate for
G3-PDH, forming a complex that spontaneously
hydrolyzes to form 3-phosphoglycerate.
o Bypassing the synthesis of and phosphate transfer from
1,3-BPG, the cell is deprived of energy usually obtained
from glycolysis and pyruvate kinase (Figure 6).
o Arsenate can also allosterically inhibit pyruvate kinase
(Figure 5).

(2) Allosteric Inhibitors

(i) Adenosine triphosphate

o ↑ ATP = ↑ energy supply

(ii) Long-Chain Fatty Acyl CoA (LCFA)

o Undergoes beta oxidation producing acetyl CoA
o ↑ LCFA = ↑ Beta oxidation activity = ↑ acetyl CoA = ↑
Krebs cycle activity = ↑ NADH, FADH2 = ↑ ATP =
↑energy supply

(iii) Acetyl CoA

o ↑ Acetyl CoA = ↑ Krebs cycle activity = ↑ NADH, Figure 6. Arsenate inhibition of glyceraldehyde 3-phosphate
FADH2 = ↑ ATP = ↑energy supply dehydrogenase.
o ↑ Acetyl CoA may also form ketone bodies and
cholesterol which, in some cases, can be disastrous

(iv) Arsenate
(B) HORMONAL REGULATION
(1) Insulin
Stimulates pyruvate kinase by dephosphorylating it
(Table 2)
Increases the synthesis of pyruvate kinase

Remember:
Insulin stimulates the synthesis of the glycolytic
enzymes but primarily hexokinase and glucokinase.

(2) Glucagon
Inhibits pyruvate kinase by phosphorylating it (Table 2)

Remember:

Table 2. Hormonal Regulation of Pyruvate Kinase

Phosphorylated Dephosphorylated
by glucagon by Insulin
Pyruvate
inactive active
kinase

Figure 5. Allosteric and hormonal regulation of pyruvate kinase.

REGULATION OF GLYCOLYSIS METABOLISM: Note #2 3 of 4

 V) APPENDIX
Figure 7. Overview of glycolysis and its highly regulated enzymes.
VI) REVIEW QUESTIONS
1) Which of the following statements about hexokinase
and glucokinase regulation is correct?
a) Glucokinase is allosterically inhibited by G6P.
b) Hexokinase is allosterically inhibited by F6P.
c) Both hexokinase and glucokinase are stimulated by
high glucose levels.
d) Glucagon inhibits glycolysis to decrease glucose
levels.
e) Insulin stimulates gluconeogenesis and inhibits
glycolysis.
2) Which of the following statements about
phosphofructokinase-1 regulation is correct?
a) ATP and citrate are allosteric inhibitors of PFK-1.
b) Fructose 2,6-bisphosphate is its most powerful
regulator.
c) Small amount of fructose 1-phosphate allosterically
activates the phosphofructokinase-2 / fructose 2,6-
bisphosphatase enzyme complex.
d) When insulin phosphorylates the
phosphofructokinase-2, it activates it.
e) When glucagon dephosphorylates fructose 2,6-
bisphosphatase, it activates it.
4 of 4 METABOLISM: Note #2
3) Which of the following statements about pyruvate
kinase is correct?
a) It is allosterically activated by fructose 1,6-
bisphosphate via feedback mechanism.
b) Glucagon phosphorylates pyruvate kinase
inactivating it.
c) Long chain fatty acids allosterically inhibit pyruvate
kinase by decreasing beta oxidation activity
d) Arsenite allosterically inhibits pyruvate kinase.
e) High acetyl CoA levels allosterically stimulates
pyruvate kinase.
4) Which of the following will occur if a person’s
glucose level is greater than 130 mL/dL?
a) Phosphofructokinase-2 / fructose 2,6-
bisphosphatase is dephosphorylated.
b) Fructose 2,6-bisphosphatase is activated.
c) Only the synthesis of pyruvate kinase is stimulated.
d) Inhibits phosphoprotein phosphatase
e) None of the above
5) During the fasting state
a) Glycolysis is favored while gluconeogenesis is
inhibited.
b) Phosphofructokinase-2 / fructose 2,6-
bisphosphatase is dephosphorylated
c) Pyruvate kinase is inhibited.
d) Glucagon activity is increased while insulin activity is
decreased.
e) Two of the choices are correct.
CHECK YOUR ANSWERS
REGULATION OF GLYCOLYSIS