1. CHAPTER 16
Glycolysis

2. 2 Pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O
Net reaction of Glycolysis
Converts: 1 Glucose
Hexose
stage
2 pyruvate
Two molecules of ATP are produced
Two molecules of NAD+ are reduced to NADH
Triose
stage
Glucose ADP NAD Pi
2 Pyruvate ATP NADH H H2O

3. 2 ATP are consumed per glucose 4 ATP are produced per glucose
Glycolysis can be divided into two stages
Hexose
stage
2 ATP are consumed
per glucose
4 ATP are produced
per glucose
Triose
stage
NET: 2 ATP produced
per glucose

4. Enzymes are needed to speed up the reactions.
Note the near equilibrium rxns
 concentration affects flux
Vs.
Those that are irreversible
 points of regulation
Hexose
stage
Triose
stage

5. Enzymes are needed to speed up The reactions.
Note the near equilibrium rxns
 concentration affects flux
Vs.
Those that are irreversible
 points of regulation
Hexose
stage
Two molecule of NADH are produced in triose
stage and are equivalent to several ATP in
terms of redox potential.
Triose
stage

6. Enzyme 1 Hexokinase
Transfers the g-phosphoryl of ATP to glucose C-6 oxygen to
generate glucose 6-phosphate
Mechanism: Mg2+ is an important cofactor, attack of C-6 hydroxyl
oxygen of glucose on the g-phosphorous of MgATP2- displacing
MgADP-
Mg2+ is an activator ion
This is an irreversible reaction

7. Figure 16.2: Enzyme 1 Hexokinase 2 lobes show induced fit model

8. Enzyme 2 Phosphoglucose Isomerase
Converts glucose 6-phosphate (an aldose) to
fructose 6-phosphate (a ketose)
Enzyme converts glucose 6-phosphate to open chain
form of fructose 6-phosphate in the active site.
Anomeric carbon
A near equilibrium reaction

9. Enzyme 3 Phosphofructokinase-1 (PFK-1)
Catalyzes transfer of a phosphoryl group from ATP to the
C-1 hydroxyl group of fructose 6-phosphate to form
fructose 1,6-bisphosphate
PFK-1 is metabolically irreversible and a critical regulatory
point for glycolysis in most cells (PFK-1 is the first committed
step of glycolysis.

10. Enzyme 4 Aldolase
Aldolase cleaves the hexose, fructose 1,6-bisphosphate
into two triose phosphates: glyceraldehyde 3-phosphate (GAP)
and dihydroxyacetone phosphate (DHAP)
This reaction is also near equilibrium
but products are favored

11. Enzyme 5 Triose Phosphate Isomerase
Conversion of dihydroxyacetone phosphate (DHAP) into glyceraldehyde 3-phosphate (GAP)
This reaction is also near equilibrium
but products are favored due to GAP metabolism

12. Fate of carbon atoms from hexose stage to triose stage
Figure 11.6
Fate of carbon atoms from the hexose stage to the triose stage of glycolysis. All numbers refer to the carbon atoms in the original glucose molecule.
Consumed 2 ATP in the
process - per one glucose
Figure 11.7

13. Enzyme 6 Glyceraldehyde 3-phosphate Dehydrogenase
Conversion of glyceraldehyde 3-phosphate (GAP) to
1,3-bisphosphoglycerate (1,3-BPG)
- One molecule of NAD+ is reduced to NADH
Pi = HPO42-
Oxidation and phosphorylation,
yielding a high energy mixed
acid hydride (NADH)
NADH can be reoxidized elsewhere, such as
the membrane associated electron transport chain

14. Enzyme 6 Glyceraldehyde 3-phosphate Dehydrogenase
The coupling of these two reactions make the overall conversion
near equilibrium
Pi = HPO42-
Very unfavorable
reaction

15. Enzyme 7 Phosphoglycerate Kinase
Uses the high-energy compound 1,3-bisphosphoglycerate
to transfer a phosphoryl group to ADP to form ATP
- 3-phosphoglycerate is formed in the process
1,3-BPG has more stored chemical energy than ATP

16. Enzyme 8 Phosphoglycerate Mutase
Catalyzes transfer of a phosphoryl group from one part of
of the substrate molecule to another
- Reaction occurs without input of ATP

17. Phosphoglycerate Mutase
Enzyme 8
Phosphoglycerate Mutase
Figure 11.7
dPGM mechanism for the conversion of 3-phosphoglycerate to 2- phosphoglycerate. (1) A lysine residue at the active site of phosphoglycerate mutase binds the carboxylate anion of 3-phosphoglycerate. A histidine residue, which is phosphorylated before the substrate binds, donates its phosphoryl group to form the 2,3-bisphosphoglycerate intermediate. (2) Rephosphorylation of the enzyme with a phosphoryl group from the C-3 position of the intermediate yields 2-phosphoglycerate.
A different phosphate is placed
on carbon-2.

18. Enzyme 9 Enolase: 2-phosphoglycerate to phosphoenolpyruvate (PEP)
Elimination of water (dehydration) yields PEP
PEP has a very high phosphoryl group transfer potential
because it exists in its unstable enol form

19. Enzyme 10 Pyruvate Kinase:
- Metabolically irreversible reaction
This reaction is another site of regulation
Pyruvate kinase is regulated both allosterically and
by covalent modification with a phosphate group
Figure 11.8
Formation of pyruvate from phosphoenolpyruvate, catalyzed by pyruvate kinase. Phosphoryl group transfer to ADP generates ATP in this metabolically irreversible reaction. The unstable enol tautomer of pyruvate is an enzyme-bound intermediate.
unstable

20. 2 glyceraldehyde 3-phosphate molecules generated
Triose Stage
2 glyceraldehyde 3-phosphate molecules generated
per one glucose
2 NADH generated
per one glucose
4 ATP generated
per one glucose

21. Table 16.1: Irreversible reactions have
large negative free energy (DG)
When DG = 0, equilibrium is established

22. Fates of pyruvate For centuries, bakeries and breweries have
exploited the conversion
of glucose to ethanol and
CO2 by glycolysis in yeast

23. Metabolism of Pyruvate
Aerobic conditions: pyruvate is oxidized to acetyl CoA
which enters the citric acid cycle for further oxidation to CO2
2. Anaerobic conditions: (microorganisms): pyruvate is converted
to ethanol
3. Anaerobic conditions: (muscles, red blood cells): pyruvate
is converted to lactate
4. Amino acid synthesis: pyruvate is a precursor to alanine

24. Anaerobic Conversion: Pyruvate to Ethanol
Yeast cells convert pyruvate to ethanol and CO2 and oxidize NADH to NAD+.
Two steps (and enzymes) are required:
Decarboxylation to form acetaldehyde by pyruvate decarboxylase.
Coenzyme thiamine pyrophosphate(TPP) is also needed
- Reduction of acetaldehyde to ethanol by alcohol dehydrogenase.
This is coupled with the oxidation of NADH
Redox is
balance

25. Reduction of Pyruvate to Lactate muscles - anaerobic
Muscles lack pyruvate dehydrogenase and cannot produce
ethanol from pyruvate
Muscle have lactate dehydrogenase to convert pyruvate
to lactate

26. Reduction of Pyruvate to Lactate muscles - anaerobic
This reaction regenerates NAD+ for
use by glyceraldehyde 3-phosphate dehydrogenase in glycolysis.
This will maintain glycolytic flux.
Lactate formed in skeletal muscles during exercise is transported to the liver via the bloodstream.
Liver lactate dehydrogenase can convert lactate to pyruvate
Lactic acidosis can result from insufficient oxygen (an increase in lactic acid and decrease in blood pH)

27. Figure 16.7 Entry of other sugars into glycolysis
Mannose and galactose are epimers of glucose
Infant mammals nurse on their mothers to drink milk, which is rich in lactose. The intestinal villi secrete the enzyme called lactase (β-D-galactosidase) to digest it. This enzyme cleaves the lactose molecule into its two subunits, the simple sugars glucose and galactose, which can be absorbed. Since lactose occurs mostly in milk, in most mammals the production of lactase gradually decreases with maturity due to a lack of constant consumption.
From ingested
fructose
From ingested
fructose

28. Figure 16.8:Fructose is converted to Glyceraldehyde 3-phosphate
Conversion requires
2 ATP molecules same
as the hexose stage
for glucose.

29. Figure 16.9: Gal-1-P is activated by attachment of a UMP.
form
Glu-6P
phosphoglucomutase

30. Regulation of Glycolysis
Enzymes that are not reversible:
Reaction 1 – hexokinase
Reaction 3 – phosphofructokinase
Reaction 10 – pyruvate kinase
These steps are metabolically irreversible, and these enzymes are regulated.
All other steps of glycolysis are near equilibrium in cells and not regulated
When ATP is needed, glycolysis is activated
- Inhibition of phosphofructokinase-1 is relieved
by accumulation of AMP or fructose 2,6-bisphosphate
- Pyruvate kinase is activated by fructose 1,6-bisphosphate
When ATP levels are sufficient, glycolysis activity decreases
- Phosphofructokinase-1 is inhibited by ATP and Citrate
- Hexokinase is inhibited by glucose 6-phosphate

31. Transporters are proteins consisting
Glucose transport into
the cell- the first step
in regulation
Transporters are proteins consisting
of ~500 AA single chain polypeptide
(Integral membrane proteins)
GLUT 2 plays a role in the regulation of insulin by the pancreas and liver-and tells liver to begin taking in glucose for storage.
Insulin binding activates GLUT 4 embedded
in plasma membrane.
Glucose uptake into skeletal and heart muscles

32. Regulation of Hexokinase
Hexokinase reaction is metabolically irreversible
Glucose 6-phosphate (product) levels increase when
glycolysis is inhibited at sites further along in the pathway
- Glucose 6-phophate inhibits hexokinase

33. Regulation of Phosphofructokinase-1 (PFK-1)
ATP is a substrate and an allosteric inhibitor of PFK-1
High concentrations of AMP (and ADP) allosterically activate
PFK-1 by relieving the ATP inhibition. ([ATP] does
not vary much in cells. [ADP] and [AMP]<< [ATP])
Elevated levels of citrate in the liver (indicate ample substrates for
citric acid cycle) also inhibit phosphofructokinase-1
ADP + ADP  ATP + AMP
Figure 16.12

34. Figure 16.13: Allosteric regulation of hexokinase and PFK

35. Glycolysis regulation in the Liver
PFK-1 by Fructose 2,6-bisphosphate (F-2,6-BP)
Fructose 2,6-bisphosphate is formed form fructose 6-phosphate
and allosterically activates PFK-1 to increase its affinity for fructose
6-phosphate.
PFK-2
PFK-1
Figure 16.14

36. Regulation of Liver Pyruvate Kinase
allosteric effectors and covalent modification
Protein
Kinase A
Alanine would indicate the plenty of protein building blocks are present for the production of macromolecules, e.g., proteins.
The hormone glucagon stimulates a protein kinase A, which
phosphorylates pyruvate kinase, converting it to a less active form

37. Assignment
Read Chapter 15
Read Chapter 16