Biochemistry class 1

Glycolysis

● aka as Embden-Meyerhof pathway or glycolytic pathway is where anaerobic degradation of

glucose to lactate with the release of energy as ATP
● It’s an example of anaerobic fermentation because chemical energy is extracted form high
energy products at the absence of Oxygen
● From this pathway only 2mols of ATP from 1mol of glucose so it’s only used in emergencies
● This pathway is important for newborn babies because they lose O2 to most parts of the
body except the brain during delivery
● This mechanism has also promoted the conservation of O2 for the brain for the survival of
brain tissue in times of stress
● The end product in the cells for glycolysis at the presence of O2 is usually pyruvate and not
lactate
● Pyruvate is then completely oxidised into CO2 and H2O in the mitochondria in TCA cycle with
the help of pyruvate dehydrogenase enzymes and enzymes of TCA within the mitochondria
● So glycolysis sets the initial footsteps in the process of aerobic oxidation of carbohydrates

● more ATP is made in complete oxidation of glucose into CO2 and H2O than in glycolysis
● While organs like the brain heavily depend on the ATP production by the complete oxidation,
where it utilises ATP made from 120g of glucose per day, some cells which lacks mitochondria
or blood supply like red blood cells and regions like cornea, lens, regions of retina depends
on glycolysis for their energy requirements
● Kidney medulla, leukocytes, testis , white muscle cells the same because they have few
mitochondria
● Tissues depending on glycolysis for energy utilises ATP made from 40g of glucose

Glucose sources
 ● starch is the storage form of glucose in plants and they have α-1,4 glycosidic linkages and
α-1,6 glycosidic branches.
● Glycogen is the glucose storage from in animal tissues and have the same linkages as above
● There are 2 types of glycogen- exogenous and endogenous
● Exogenous is glycogen we obtain from animal products and Endogenous means glycogen
synthesised and stored in our tissues
● Exogenous starch/glycogen is hydrolysed in the intestinal tract to glucose and the
endogenous glycogen is converted into glucose or glucose-6-phosphate by the enzymes in
the cells
● Disaccharides like milk and sugar (lactose and sucrose) is an important source
● Glucose absorbed by the intestinal cells goes through the portal vein and then to the general
circulation for the other tissues to use
● Liver is the 1st organ to remove glucose from the portal vein
● At high levels of glucose in blood, liver take the glucose and use it for glycolysis and
glycogenesis
● At low blood glucose levels the liver beaks down the glycogen into glucose in
gluconeogenesis and glyconeogenesis
● Liver is also the organ which receives the blood with highest levels of glucagon and insulin

Glucose metabolism in different cells

independent
↓insulin
● in RBCs - mainly by glycolysis. Transport is catalysed by glucoe transporter GLUT-1. No
mitochondria so end product is lactic acid.NADPH is made in RBCs via utilisation of glucose
in the Pentose pathway to keep glutathione in reduced state which is important in the
destruction of peroxides and H2O2 (peroxides cause irreversible damage to membranes,
DNA etc)
● In Brian cells - takes glucose via facilitated transport with the help of GULT-3 in an insulin
dependent way. Glycolysis gives pyruvate which is oxidised. The pentose pathway is active in
brain cells so same function as RBCs
● Muscle and heart cells - insulin stimulate transport of glucose into cells via GLUT-4. GLUT-4
is located in intracellular vesicles and cannot facilitate glucose transport without insulin
because insulin induced a signal cascade where the transporter containing vesicle is
translocated and fused with the membrane where is can facilitate the glucose transport.
 Glucose is completely oxidised to produce ATP. These cells synthesise significant amounts of
glycogen which is stored in tissues for future use.
● Adipose cells - as with muscle cells, there cells require insulin for glucose transport into GLUT-4

cells. Pyruvate is made from glucose and is oxidised by the pyruvate dehydrogenase complex
to give A-CO-A which is primarily used for de novo fatty acid synthesis. Glycolysis also
provide C for glycerol-3-phosphate which is needed for the synthesis of triglycerol. Adipose
cells can also perform glycogenesis and glycogenolysis but it limited in comparison to heart,
muscle and liver cells
● Liver cells - have the greatest number of ways to utilise glucose. Glucose uptake done by
insulin independent transporter GLUT-2 which is low affinity and high capacity. Pentose
pathway is present and NADPH is made for maintnece of reduced glutathione and for many
reaction catalysed by ER enzymes. Pentose pathway is also important to produce the pentose
sugar part of some nucleotides like DNA, RNA and ATP. Live also stores glucose as glycogen.
Liver cells both does complete oxidation of pyruvate and fatty acid synthesis after glycolysis.
It makes triglyerols during the process of making VLDLs. The liver also covert 3C products
like lactate, pyruvate, glycerol and alanine into glucose by gluconeogeneis for other cells and
brain cell’s energy requirements.

Glycolysis pathway

Cells normally use about 30 steps to convert glucose into CO2 and H2O. Cells produce ATP by the
‘controlled’ burning of glucose.

Glycolysis has 3 main stages

1. Priming stage
fructose1,6-bisphosphate* 2Axp 2H
+
2ATp4 -
+

+ D
D.
glucose

2. Splitting stage

D fructose 16 bisphosphate" - 2 (G3P)

D-glyceraldehyde 3-phosphate

3. Oxidoreduction - phosphorylation stage

+ 2H*- 2 Lactate
+ 4ATP" 2H2 +

+4ADPS +2P;
2
D-Glyceraldehyde 3-phosphate

Sum of reactions:

2ATP" +2H20
+2ADP"+2p;->2 [lactate +
D.
glucose
 – The priming stage involves in converting D-glucose into D-fructose-1,6-bisphosphate with
the help of 2 ATP
– The ATP is not spent because ATP is regained in the latter steps
– The D-fructose-1,6-bisphosphate is split into 2 D-glyceraldehehyde 3-phosphate in splitting
stage
– In oxidoreduction-phosphorylation stage the D-glyceraldehyde 3-phosphate molecules are
converted into L- lactate and gives 2 molecules of ATP from 1 glucose molecule.

Priming of glucose

Even though ATP is consumed, converting glucose into glucose-6-phosphate make the molecule
charged and hydrophilic because they are converted into phosphate esters. This makes them
impermeable to cell membrane and traps it in the cell
This reaction is thermodynamically favourable and therefore and irreversible reaction.
done Hexokinase (Liver)
by or
glucokinase

Glucose-6-phosphate is then converted into fructose-6-phosphate by phospoglucose isomerase.

This reaction is reversible and has no regulation

Fructose-6-phosphate is converted into fructose 1,6 bisphosphate by 6- phospofructo-1-kinase.

This reaction is ATP dependent. This reaction is regulated by several efffectors and this enzyme is
the key regulatory enzyme of Glycolysis.
fructose 16 bisphosphate
Irreversible reaction *
Key regulatory reaction] fructose-6-phosphate
-

6. Phosphofructo-1- kinase
glycolysis
->
of Regulatory enzyme

Splitting of s phosphorylased intermediate

1. Fructose 1,6 bisphosphate di hydroxy acetone phosphate / DHAP + glyceraldehyde-3-

phosphate /GAP (a3P)

Enzyme : fructose 1-6 bisphosphate aldolase

Reaction is reversible (aldole cleavage/ condensation)

2. DHAP GAP (a3P)

Triose phosphate isomerase inter conversion of DHAP and GAP / convert both into one type

Now ⸻> 2 molecules of GAP is made from 1 molecule of glucose

Oxidoreduction reactions and ATP synthesis

Enzyme : glyceraldehyde-3-phosphate dehydrogenase

Aldehyde ⸺ oxidation⸺> carboxylic acid

NAD+ ⸺ reduction ⸻> NADH

GAP
M
1,3- bisphospoglycerate

1,3- bisphospoglycerate is a mix anhydride of carboxylic and Phosporic acid.

It has a large amount of - free energy and often participate in reactions which produce ATP

exergonic ( ΔH - ) reaction and an endergonic ( ΔH + ) reaction

Exergonic - oxidation
Endergonic - reduction

2nd endergonic reaction is when the carboxylic acid and phosphoric acid makes a mix anhydride

Overall sum of reactions give a ΔG of +1.5kJmol

This reaction is freely reversible in cells

Catalytic mechanism of glyceride-3-phosphate dehydrogenase

 -> Thiolester
OX

-> NADH

Red

1. G3P reacts with —SH group of a cystine residue of the enzyme. This forms a thiohemiacetal
2. Internal RedOx occurs - thiohemiacetal is oxidised into thioester while NAD+ is reduced into
NADH
3. Thiol ester reacts with Pi to make the mixed anhydride. This will regenerate the ⸺SH group.
4. Mixed anhydride disassociate from enzyme and NAD+ replace NADH
5. The -COOH is not made from -CHO but from the high energy thiol ester, this -COOH is the
one which reacts with Pi to make the mixed anhydride
6. This reaction is coupled with the reduction of NAD+ so regeneration of NAD+ should occur to
continue glycolysis

ATP synthesis

ATP is made from 1,3-bisphospoglycerate

Enzyme : phosphoglycerate kinase

this is the first site of ATP production in glycolysis

2 molecules of ATP made from 1 glucose molecule.
The ATP used to convert glucose into glucose-6-phosphate is reimbursed.

the glyceraldehide 3 phosphate dehydrogenase- phospoglycerate kinase system is an example

of substrate level phosphorylation where the substrate participates in enzyme catalysed reactions
that yields ATP or GTP

This is contrasting to oxidative phosphorylation catalysed by mitochondrial ETC and ATP synthase

The 2 enzymes glyceraldehide-3-phosphate dehydrogenase and phopspglycerate kinase couples

oxidation with a phosphorylation without the involvement of a membrane system.

Phospoglycerate mutase enzyme

Enzyme catalyse :
3-phospoglycerate 2-phospoglycerate

● Active site intermediate: 2,3 bisphospoglycerate - obligatory

● Freely reversible reaction

Intermediate is important to induce the catalytic activity because it is needed to generate the E-P

● this enzyme is bi functional, it act as a mutase to make 2,3 bisphospoglycerate and can also
act as a phosphatase which hydrolyses 2,3 bisphospoglycerate to make 3- phospoglycerate
and Pi
● All cells have small amounts of 2,3 bisphospoglycerate to make the E-P complex but the
RBCs have a significantly larger amount because it acts a an allosteric regulator of the
 binding of oxygen to Hb.
● In RBCs 10-25% of glucose is converted into lactate by the way of BPG shunt for the
synthesis of 2,3 bisphospoglycerate
● The BPG shunt bypasses the phospoglycerate kinase / PGK step so no ATP made via the BPG
shunt

2,3 Bisphospoglycerate in RBCs

● they are hydrolysed by multiple inositol polyphosphate phosphatases / MIP phosphate

● They convert 2,3 bisphospoglycerate into 2-phospoglycerate not 3, and expands the BPG
shunt to by pass the reaction catalysed by phospoglycerate mutase
● activity of MIP phosphatase set the C of 2,3BPG ; the allosteric effector of Hb.

Fate of 2-phospoglycerate

● enolase enzyme eliminates water form 2PG to make Phospoenolpyruvate/ PEP

● This reaction generates a higher energy level of the exuded lower one (hydrolysis ΔG for PEP
is more negative than 2PG)
● Pyruvate kinase does another substrate level phosphorylation. This is not reversible under
intercellular conditions

Final step of glycolysis

● A freely Oxidoreduction reaction

● This is catalysed by lactate dehydrogenase

Pyruvate ⸺reduction⸺> L-lactate

NADH ⸻ oxidation ⸻> NAD+

L-lactate is only produced in this forward reaction

Utilisation can be only done in reverse reaction

so Lactate dehydrogenase is responsible for both production and the utilisation of L-lactate

Summary
 ⑧

ATP Yield and Balanced Equation for Anaerobic Glycolysis

● 1 glucose molecule is converted into 2 molecules of lactate

● 2 ATP molecules are utilised for priming glucose
● Subsequent steps yield 4 molecules of ATP
● Net gain of ATP is 2

[ D-Glucose +2Axp + 2P,- 2-[Lactate + 2AT"+ 2Hz ]

● cells have a limited amount of ADP and Pi so the flux though glycolysis depends on the
adequate amount of these substrates
● If ATP is not utilised for work ATP generation through glycolysis halts
● And ATP generated by glycolysis must be utilised in work

ATP4- + H2O ⸺> ADP 3- + Pi2- + H+ + work

Overall reaction for 1 molecule of glucose with the addition of the use of ATP for work,
(Add the 2 equations)
D-glucose ⸺> 2 L-lactate + 2H+

anaerobic glycolysis cause the generating of lactic acid which is a problem because it changes
the intracellular Ph and intracellular Ph should be maintained at its optimal parameters for optimum
enzyme activity

NADH Generated by Glycolysis Has to Be Oxidized Back to NAD+: Role of Lactate

Dehydrogenase and Substrate Shuttles

Anaerobic glycolysis

● NAD+ and NADH do not appear in the final balanced equations for glycolysis as the
generation and the utilisation of NADH is balanced in the glycolysis pathway
● 2 molecules of NADH are made by glyceraldehyde-3-phosphate dehydrogenase
● The formed NADH molecules are utilised by lactate dehydrogenase

This 2 reactions should occur perfectly under anaerobic or mitochondria lacking contains for
glycolysis to occur smoothly