Glycolysis (Glycolytic Pathway)
- 1st pathway in carbohydrate/glucose metabolism; where glucose first enters
- Tissues that synthesize glucose: liver, kidney (prioritized to synthesize glucose)
o But if concentration is low, glucose consumption is not prioritized
o Role is to supply glucose to the blood stream
- Tissues that use glucose as their primary source of energy: brain and nervous tissue, muscle, erythrocyte, testes
Essential Features of Glycolysis
• also called the Embden-Meyerhof (or Warburg) Pathway
• glycolysis is a catabolic pathway, glucose enters here and supposed to be broken down
• Total energy release is only 5%, because after the pathway of glucose, product of breakdown goes into
another pathway (where rest of energy is produced upon breakdown of the particular intermediate or
product coming from glycolysis)
• coupled reactions involving ATP hydrolysis are also used to drive the glycolytic pathway
• we expect to be energy producing, net yield of ATP. But there are some steps in glycolysis that also would
lead ATP as possible source of energy
• Considered to be a universal pathway because eessentially all cells are capable of undergoing glycolysis
• There are 10 reactions in glycolysis
• First phase: glucose converted to two triosephosphate molecules (G-3-P)
• Glyceraldehyde-3-phosphate (G-3-P)
• Second phase: produces two pyruvate molecules, for every molecule of glucose
• Products of glycolysis are pyruvate, ATP and NADH
• Three possible fates for pyruvate
Glycolytic Pathway Showing all 10 steps:
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There are parts of the glycolysis that are considered to be catalyzed by regulatory enzymes
These are the points in glycolysis that are subject to regulation (Step 1, Step 3, and Step 10)
The rest of the steps are not subject to regulation because it is a common enzyme to the opposite pathway (which is
gluconeogenesis)
o Cannot be subject to regulation because you will end up having a futile pathway
o Activating or inhibiting it will just activate or inhibit the forward and backward reaction.
There are some steps that are ATP consuming (Step 1 and Step 3)
We expect a net yield of ATP in glycolysis, we expect it to form in Step 7 and Step 10
NADH is also formed in Step 6
o NADH is located in the cytosolic side of the cell; Glycolysis occurs in the cytosol
o For step 6 to continue, you must have a steady supply of NAD+
 To have a steady supply of NAD+, there must be a way to recycle NADH to NAD+
o If this occurs in the matrix then there is no problem, because all of NADH just have to enter and give
electron to Complex 1 for it to be oxidized
o But it does not happen here because NADH is not directly connected to Complex 1 since it is in the cytosolic
side.
o So to recycle NADH to NAD+:
 Under aerobic condition: NADH is recycled thru shuttles (glycerol phosphate shuttle or malate
aspartate shuttle). So the electrons are passed on to certain carriers and these electrons are brought
into the mitochondria.
 For malate aspartate shuttle, receiver on other side is complex 1; For glycerol phosphate shuttle
receiver on the other side is complex 2)
 We are able to recycle NADH to NAD+ and at the same time synthesize ATP
 Under anerobic condition: recycled thru the production of lactate
3 Fates of Pyruvate from Glycolysis
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Under aerobic condition, pyruvate is converted to Acetyl-CoA.
o Acetyl-CoA enters tricarboxylic acid cycle, which is connected to the electron transport chain, and which
is connected to oxidative phosphorylation, further producing ATP
o Glucose to pyruvate only represents 5% of the amount of energy released from glucose
o The rest will be released thru conversion of acetyl-coA, then entering the TCA cycle
Under anaerobic condition, pyruvate is converted to lactate in animal and human cells
Under anaerobic condition, pyruvate is converted to ethanol and carbon dioxide in yeast cells
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Anaerobic conditions does not produce ATP, the conversion is due to the recycling of NADH back to NAD+; there
are no shuttles
Step 1: Conversion of Glucose to Glucose-6-phosphate
 It is a combination of two reactions (coupled reaction)
o First: endergonic conversion of glucose to glucose-6-phosphate
o Second: exergonic hydrolysis of ATP
 This is an ATP consuming step
 To count ATP: -1 ATP
 ATP is not the only source of energy but also the source of phosphate group
 It is a regulatory enzyme (main point of regulation)
o Hexokinase and glucokinase
 There is Magnesium as cofactor
o Whenever ATP is involved as a substrate, there is usually a divalent cation. Role is to stabilize ATP, serves
as the electron sink
 It is an irreversible reaction, which is subject to regulation
o Subject to regulation because the other pathway does not have this
 Advantage of the Conversion of Glucose to Glucose-6-phosphate: when glucose is converted to glucose-6phosphate, it is imparted a negative charge. So it makes it difficult for glucose to get out of the cell, which traps
glucose inside the cell so that it can enter the metabolic pathways.
 Enzymes: the isozymes, hexokinase and glucokinase, catalyzes this step
o Glucokinase (10mM) has a higher Km compared to hexokinase (0.10 mM)
o Higher Km tells us that glucokinase can only consume glucose when the cell is rich in glucose
o The concentration of glucose in a cell is 4mM
 Glucokinase is higher so it requires a much higher concentration before glucokinase in the liver
can consume the glucose. Only turns on when the cell is rich in glucose
 With low Km of hexokinase, hexokinase is always active
o Hexokinase and glucokinase is a regulatory enzyme that is subject to regulation
Step 2: Conversion of glucose-6-phosphate to fructose-6-phosphate
 The glucose-6-phosphate isomerase enzyme that catalyzes is not an enzyme due to
regulation since its common to both glycolysis and gluconeogenesis
 Isomerization reaction: Aldohexose to ketohexose
 No net reaction
Step 3: Conversion of fructose-6-phosphate to fructose-1,6-bisphosphate
 ATP consuming step, coupled reaction
 Reaction is the combination of endergonic conversion of fructose-6-phosphate to fructose-1,6-bisphosphate, and
exergonic hydrolysis of ATP
 ATP is source of energy and source of phosphate group for the product
 To count ATP: -1 ATP
 Enzyme: PFK-1 (Phosphofructokinase) is the main regulatory enzyme for glycolysis
 Product is fructose-1,6-bisphosphate, which is a key intermediate
 Mechanism also calls for magnesium, since ATP is part of the substrate
 PFK1 catalyzes the committed step in glycolysis
o Will lead to the formation of the intermediate, fructose-1-6-biphosphate
o Ince intermediate if formed, no turning back, will proceed toward glycolysis
 PFK1 is highly regulated
Being a catabolic enzyme, we expect ATP to inhibit that because plenty of ATP is an indicator that the cell
in is the state of high energy charge
AMP or ADP can activate PFK1, it tells PFK1 that ATP of cells are already depleted so it can go on and produce
more ATP
Citrate is also an allosteric inhibitor
Fructose-2,6-bisphosphate is allosteric activator, activate enzyme, level of this is highly regulated
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Step 4: Splitting of fructose-1-6-biphosphate to DHAP and G-3-D
 Not subject to regulation
 Splitting of fructose-1-6-biphosphate to two types of triosphosphate:
o Dihydroxyacetone phosphate (DHAP) is a ketotriose
o D-glyceraldehyde-3-phosphate (G-3-D ) is an aldotriose
o They are isomers
 Aldolase is the enzyme
 Reversible reaction
 Not subject to regulation
 Dihydroxyacetone phosphate
Step 5: Conversion of DHAP to G-3-D
 Dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (G-3-D) are isomers
 Dihydroxyacetone phosphate (DHAP) is converted to glyceraldehyde-3-phosphate
 The rest of the steps must be multiplied to two to get a net production because there
are two G-3-D
o One coming from fructose-1-6-biphosphate and other coming from DHAP
Step 6: Oxidation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate
 This reaction involves addition of a phosphate group, as well as an electron
transfer (oxidation of carbon 1) which is aldehyde to carboxylic acid
 Electron receiver is NAD+ which is reduced to NADH
 Enzyme: glyceraldehyde-3-phosphate dehydrogenase, not subject to
regulation, suppose to catalyze a reversible reaction. Common to opposing pathway
 NADH is produced in glycolysis
 Under aerobic condition: NADH can be recycled via glycerol phosphate shuttle or malate aspartate shuttle. This
is good because we have recycled NADH back to NAD+ and make ATP at the same time via oxidative
phosphorylation.
 Under anaerobic condition: will be done through probation of lactate or ethanol or carbon dioxide along cells
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Oxidize aldehyde to carboxylic acid first, then add phosphate group
Step 7: 1,3-bisphosphoglycerate is converted to 3-phosphoglycerate
 Not subject to regulation because it is part of gluconeogenesis
 1,3-bisphosphoglycerate is a high energy compound so cells will take
advantage of that
 If 1,3-bisphosphoglycerate is converted to 3-phosphoglycerate, it gives
energy as much as -11.8 kcal/mol
 Production of ADP required +7.3 kcal/mol, so we have more than enough
 We are able to make ATP; production of ATP that has nothing to do with ETC or OP
 Synthesis of ATP is via substrate level phosphorylation
 Enzyme: phosphoglycerate kinase (not for regulation)
 To count ATP: +2 ATP
o Because you produce ATP, and after step 5, there is times two, since two G-3-P
Step 8: 3-phosphoglycerate to 2-phosphoglycerate
 isomerization of 3-phosphoglycerate to 2-phosphoglycerate
 Enzyme: reaction is catalyzed by phosphoglyceromutase
 common to both glycolysis and gluconeogenesis
Step 9: 2-phosphoglycerate to phosphenolpyruvate
 2-phosphoglycerate loses one molecule of water, producing phosphenolpyruvate (water release)
 dehydration reaction
 Enzyme: Enolase catalyzes the reaction and requires a Mg2+ cofactor
 Not subject for regulation, common to gluconeogenesis
 Phosphoenolpyruvate contains a high energy bond
Step 10: Phosphenolpyruvate (PEP) to pyruvate and ATP
 Phosphoenolpyruvate is a high energy compound, releases an energy of -14.9 when converted to pyruvate
o Cells will take advantage of that to synthesize ATP via substrate level phosphorylation
o G of hydrolysis of PEP is more than that of ATP
 Phosphenolpyruvate (PEP) transfers its phosphate group to ADP,
producing ATP and pyruvate
 To count ATP: +2 ATP
 Irreversible reaction
 Enzyme: Reaction is catalyzed by pyruvate kinase
 This is the third point of regulation for glycolysis
 PK is allosterically activated by AMP, F-1,6-bisP
o Due to energy charge, low E.C. so it should go on and activate the catabolic pathway
o Positive forward activation because F-1,6-bisP is formed before that
 PK is allosterically inhibited by ATP and acetyl-CoA
o Plenty of acetyl-coA that was formed from pyruvate
 In the final stages of glycolysis, two molecules of pyruvate are produced for each molecule of glucose that entered
the pathway
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dG for sum is actually positive, but if concentrations of metabolites are considered the dG are mostly negative.
Net dG = -98 (exergonic)
Example: glucose -> 2 pyruvate
IN
Step 1: ATP
Step 3: ATP
Step 6: 2 Pi + 2 NAD+
Step 7: 2 ADP
Step 9:
Step 10: 2ADP
OUT
ADP
ADP
2 NADH, 2H+
2 ATP
2 H2O
2 ATP
Overall reaction: glucose + 2ADP + 2 Pi + 2NAD+ -> 2 pyruvate + 2 ATP + 2H2O + 2NADH + 2H+
SUMMARY:
 glucose enters glycolysis, first part uses ATPm but second are ATP generating steps
 net will be glucose -> 2 pyruvate (2ADP -> 2ATP) and (2NAD+ -> 2NADH)
 pyruvate is converted to lactate for animal cells, converted to ehtnaol in yeast cells
Step 11: Anaerobic Metabolism of Pyruvate
 under anaerobic condition, pyruvate is converted to lactate, catalyzed by the enzyme lactate dehydrogenase
 catalyzes a reversible reaction
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purpose is to recycle NADH back to NAD+
Lactate dehydrogenase (LDH) is a tetrameric isoenzyme consisting of H and M subunits;
o H4 predominates in heart muscle, and M4 in skeletal muscle
LDH is supposed to exist in terms of isozymes, difference is which direction will be favored
Step 11: Alcoholic Fermnetation
 For the case of yeast cells, formation of ethanol is because of recycling of NADH back to NAD+
 In some organisms, pyruvate is converted to ethanol in a process requiring thiamine pyrophosphate as a coenzyme
 Part 1: Decarboxylation of pyruvate to acetaldehyde
 Part 2: Reduction of acetaldehyde to ethanol
 Pyruvate decarboxylase is the enzyme that catalyzes the first reaction
 this enzyme require Mg2+ and the cofactor, thiamine pyrophosphate (TPP).
 Alcohol dehydrogenase catalyzes the conversion of acetaldehyde ethanol
OTHER HEXOSES:
 Other hexoses can serve as substrates for glycolysis
 They have different entry points, these hexoses are, fructose, mannose and
galactose
 Mannose is converted to mannose-6-phosphate and enters the glycolysis as
fructose-6-phosphate so it enters via the second step and eventually become
fructose-1-6-bisphosphate
 Fructose will become fructose-1-phosphate and enters via
dihydroxyacetonephosphate an glyceraldhyde-3-phosphate
 Galactose does not directly enter glycolysis but first it is converted to glucose,
o Galactose converted to galsctose-1-P and then UDP-Glucose and then glucose-1-P, then it become
glucose-6-P
Glycerol can enter glycolysis
 Glycerol can also enter glycolysis and can also serve as a possible
subrstrate for glycolysis
 Glycerol enter first is converted to glycerol-3-P, then it eventually
thru oxidation of Carbon 2, become dyhydroxyacetone phosphate
wherein NADH is the receiver of electrons.
Example: glucose -> 2 lactate (anerobic conditions)
IN
Step 1: ATP
Step 3: ATP
Step 6: 2 Pi + 2 NAD+
Step 7: 2 ADP
Step 9:
Step 10: 2ADP
Step 11: 2 NADH + 2 H+
OUT
ADP
ADP
2 NADH, 2H+
2 ATP
2 H2O
2 ATP
NADP+
You can cancel out:
Overall reaction: glucose + 2ADP + 2 Pi -> 2 lactate + 2 ATP + 2H2O