1. Lecture 1
Glycolysis
glykus sweet; lysis loosen

2. Louis Pasteur’s investigation into fermentation of grape
Louis Pasteur’s scientific investigations into fermentation of grape sugar were pioneering studies of glycolysis.
Louis Pasteur’s investigation into fermentation of grape

3. Brief History of Glycolysis
1897 : Hans and Eduard Buchner report the discovery of cell-free fermentation using yeast
1901 : Wroblewski discovered that inorganic phosphate stimulated fermentation.
1903 : Buchner, Buchner and Hann confirmed the phosphate effect.
1906 : Harden and Young reported on the phosphate effect.

4. An English biochemist. He shared the Nobel prize in chemistry in 1929 with Hars Kral August simon von Eulerchelpin for their investigations into the fermentation of sugar and fermentative enzymes.
Sir Arthur Harden
(12 October 1865 – 17 June 1940)

5. A: 25 mls of glucose + 25 mls of yeast juice
Harden and Young’s famous phosphate experiment, published in 1906
A: 25 mls of glucose + 25 mls of yeast juice
B: 25 mls of glucose + 25 mls of yeast juice + 5mls of 0.3M phosphate
C: As in B but at 70 min a second phosphate addition was made.

6. The Harden and Young ester
(frucutose 1,6-bisphosphate, sugar di-phosphate)
Harden and Young demonstrated that the inorganic phosphate disappeared from solution
They suggested that the phosphate was converted to a glucose phosphate ester.
In 1906 Ivanov isolated a phosphate ester in the form of its copper salt.
He correctly determined the empirical formula as C3H5O2PO4H2, naming it a triose phosphate.
However, it was not until 1928 that it was identified as fructose -1,6-biphosphate.

7. Discovery of glucose phosphate
In 1914, Harden and Robinson detected a hexose monophosphate.
Not until 1931 was this compound identified as glucose-6-phosphate.
Slowly but surely all the main reactants of glycolysis were identified.
Through the use of metabolic inhibitors it was possible to work out the sequence of reactions.

8. How does one work out the sequence of reactants?
Consider a pathway in which compound A is converted to compound E.
Compounds B, C and D are isolated from the pathway, but their sequence is unknown.
An inhibitor which blocks this pathway, prevents the utilization of compound A and causes the accumulation of compounds A and B.
There is no accumulation of C,D or E.

9. How does one work out the sequence of reactants?
A second inhibitor, which also blocks the formation of E, results in the accumulation of A, B and D.
No accumulation of C or E is observed.
Can you predict the sequence of the pathway?
A  B  D  C  E

10. How does one work out the sequence of reactants?
Now that we have radiotracers we could use another approach:-
If we start with radioactive glucose we can add this to a culture of anaerobic yeast and identify the intermediates that are labelled
This would require a method to separate and identify the intermediates:
The pathway of photosynthesis was worked out by this method starting from 14C-labelled CO2.

11. Characteristics of Glycolysis
Glycolysis does not require O2, but it can occur under aerobic conditions too.
The earliest organisms were thought to be anaerobic, so glycolysis probably preceded aerobic respiration
Photosynthetic bacteria e.g. conducted photosynthesis using reduced substances like H2S as their source of reducing power: H2S  S

12. Characteristics of Glycolysis
Once the capacity to use H2O evolved, O2 was produced as the byproduct rather than S.
H2O  O2
Aerobic respiration then became a possibility
Two experimental systems became common for studying glycolysis, namely yeast (fermentation) and muscle tissue (glycolysis).
Eventually, it became obvious that the two pathways were essentially identical.

13. Characteristics of Glycolysis
in yeast (fermentation) :
glucose  pyruvate  ethanol
In muscle (anaerobic respiration) :
glucose  pyruvate  lactate
In both systems (and in all organisms) ten enzymes are involved in converting glucose to pyruvate.

14. yeast Gluconeogensis 1 ATP 2 Anaerobic Aerobic muscle Alcoholic
Fermentation
Gluconeogensis 1
ATP
ATP 2
Anaerobic
Aerobic
Lactic acid
Fermentation
muscle

16. Glycolysis
p. 579

17. ACTIVE FIGURE 18. 1 The glycolytic pathway
ACTIVE FIGURE 18.1 The glycolytic pathway. Test yourself on the concepts in this figure at
Fig. 18-1, p. 580

18. FIGURE 18.2 In the first phase of glycolysis, five reactions convert a molecule of glucose to two molecules of glyceraldehyde-3-phosphate.
Fig. 18-2, p. 581

19. Characteristics of Glycolysis

20. Details of Glycolysis 1. Location of the pathway (cytoplasm)
2. Pathway of carbon reactions.
3. Location of redox reactions.
4. ATP production and energy balance.
5. The need to regenerate NAD+.
6. Regulation of the pathway.

23. Glycolysis

27. The overall reaction of glycolysis which occurs in the cytoplasm is represented simply as:
C6H12O6 + 2 NAD+ + 2 ADP + 2 P -----> 2 pyruvic acid, (CH3(C=O)COOH + 2 ATP + 2 NADH + 2 H+
glucose with six carbons is converted into two pyruvic acid molecules with three carbons each. Only a net "visible" 2 ATP are produced from glycolysis.

28. Reaction 1: Phosphate Ester Synthesis
Phosphate is added to the glucose at the C-6 position. The reaction is a phosphate ester synthesis using the alcohol on the glucose and a phosphate from ATP.
This first reaction is endothermic and thus requires energy from a coupled reaction with ATP. ATP is used by being hydrolyzed to ADP and phosphate giving off energy and the phosphate for reaction with the glucose for a net loss of ATP in the overall glycolysis pathway.
Hydrolysis: ATP + H2O --> ADP + P + energy
P = PO4-3; ATP = adenine triphosphate;
ADP = adenine diphosphate
This reaction is catalyzed by hexokinase.

29. Priming reactions in cycle
FIGURE 18.3 Just as a water pump must be “primed” with water to get more water out, the glycolytic pathway is primed with ATP in steps 1 and 3 in order to achieve net production of ATP in the second phase of the pathway.

35. Substrate-induced cleft closing = General feature of kinases
Large
Conformational
Change
Induced-fit Model
* Environment  nonpolar
* Closing the cleft  Water away
Substrate-induced cleft closing
= General feature of kinases

36. Glucose is kept in the cytoplasm upon phosphorylation
ANIMATED FIGURE 18.4 Phosphorylation of glucose to glucose- 6-phosphate by ATP creates a charged molecule that cannot easily cross the plasma membrane. See this figure animated at brookscole.com/ggb3

37. Glucose-6-phosphate is the branch point for several
metabolic pathway

38. Reaction 2: Isomerization
The glucose-6-phosphate is changed into an isomer, fructose-6-phosphate. This means that the number of atoms is unchanged, but their positions have changed.
This works because the ring forms may open to the chain form, and then the aldehyde group on glucose is transformed to the keone group on fructose. The ring then closes to form the fructose-6-phosphate.
This reaction is catalyzed by
phosphoglucoisomerase.

41. Formation of enol

43. Reaction 3: Phosphate ester synthesis The second priming reaction
This reaction is virtually identical to reaction 1 The fructosee-6-phosphate has an alcohol group on C-1 that is reacted with phosphate from ATP to make the phosphate ester on C-1.
Again this reaction is endothermic and thus requires energy from a coupled reaction with ATP. ATP is used by being hydrolyzed to ADP and phosphate giving off energy and the phosphate for reaction with the glucose for a net loss of ATP in the overall glycolysis pathway.
Hydrolysis: ATP + H2O --> ADP + P + energy
This reaction is catalyzed by phosphofructokinase.
Fructose-1,6-bisphosphate

47. At high ATP concentration, phosphofructokinase (PFK) behaves cooperatively and the plot of enzyme activity versus [fructose-6-phosphate] is sigmoid. High [ATP] thus inhibits PFK, decreasing the enzyme’s affinity for fructose-6-phosphate
FIGURE 18.8 At high [ATP], phosphofructokinase (PFK) behaves cooperatively and the plot of enzyme activity versus [fructose-6-phosphate] is sigmoid. High [ATP] thus inhibits PFK, decreasing the enzyme’s affinity for fructose-6-phosphate.

53. Reaction 4: Split Molecule in half
Fructose-1,6-bisphosphate
The six carbon fructose diphophate is spit into two three-carbon compounds, an aldehyde and a ketone.
The slit is made between the C-3 and C-4 of the fructose. The ring also opens at the anomeric carbon. The product on the right is the glyceraldehyde.
Technically this is called a reverse aldol condensation.
This reaction is catalyzed by aldolase.

58. Aldol cleavage

59. The chemical evidence for the schiff base intermediate in class I aldolase

60. Reaction 5: Isomerization
The dihydroxyacetone phosphate must be converted to glyceraldehyde-3-phosphate to continue the glycolysis reactions. This reaction is an isomerization between the keone group and an aldehyde group.
As a result of this reaction, all of the remaining glycolysis reactions are carried out a second time. The first series of reactions occurs with the first glyceraldehyde molecule from the orginal split. Then the second series of reactions occurs after the isomerization of the dihydroxyacetone into the glyceraldehyde.
This reaction is catalyzed by triose phosphate isomerase.

64. Mechanism: Triose phosphate iomerase salvages a three-carbon fragment
1. Kinetically perfect enzyme
2. Loop as a lid to shut enediol intermediate

65. Methyl glyoxal pathway
This pathway does not produce any ATP, this pathway does not replace glycolysis it runs simultaneously to glycolysis and is only initiated with an increased concentration of sugar phosphates. One believed purpose of the methylglyoxal pathway is to help release the stress of elevated sugar phosphate concentration. Also when methylglyoxal is formed from DHAP, an inorganic phosphate is given off which can be used to replenish a low concentration of needed inorganic phosphate. The methylglyoxal pathway is a rather dangerous tactic, both because less energy is produced and a toxic compound, methylglyoxal is formed