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UNIT- II
Glycolysis
Glycolysis, which translates to "splitting sugars", is the process of releasing
energy within sugars. In glycolysis, a six-carbon sugar known as glucose is split
into two molecules of a three-carbon sugar called pyruvate. This multistep
process yields two ATP molecules containing free energy, two pyruvate
molecules, two high energy, electron-carrying molecules of NADH, and two
molecules of water.
Definition
Glycolysis can be defined as the sequence of reactions for the breakdown of
Glucose (6-carbon molecule) to two molecules of pyruvic acid (3-carbon
molecule) under aerobic conditions; or lactate under anaerobic conditions along
with the production of small amount of energy.
Glycolysis
Glycolysis is the process of breaking down glucose.
Glycolysis can take place with or without oxygen.
Glycolysis produces two molecules of pyruvate, two molecules of ATP,
two molecules of NADH, and two molecules of water.
Glycolysis takes place in the cytoplasm.
There are 10 enzymes involved in breaking down sugar. The 10 steps of
glycolysis are organized by the order in which specific enzymes act upon
the system.
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Figure 1 Glycolysis Cycle
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Glycolysis can occur with or without oxygen. In the presence of oxygen,
glycolysis is the first stage of cellular respiration. In the absence of oxygen,
glycolysis allows cells to make small amounts of ATP through a process of
fermentation.
Glycolysis takes place in the cytosol of the cell's cytoplasm. A net of two ATP
molecules are produced through glycolysis (two are used during the process and
four are produced). Following are the 10 steps of glycolysis:
Step 1
The enzyme hexokinase phosphorylates or adds a phosphate group to glucose
in a cell's cytoplasm. In the process, a phosphate group from ATP is transferred
to glucose producing glucose 6-phosphate or G6P. One molecule of ATP is
consumed during this phase.
Step 2
The enzyme phosphoglucomutase isomerizes G6P into its isomer fructose 6-
phosphate or F6P. Isomers have the same molecular formula as each other but
different atomic arrangements.
Step 3
The kinase phosphofructokinase uses another ATP molecule to transfer a
phosphate group to F6P in order to form fructose 1,6-bisphosphate or FBP. Two
ATP molecules have been used so far.
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Step 4
The enzyme aldolase splits fructose 1,6-bisphosphate into a ketone and an
aldehyde molecule. These sugars, dihydroxyacetone phosphate (DHAP) and
glyceraldehyde 3-phosphate (GAP), are isomers of each other.
Step 5
The enzyme triose-phosphate isomerase rapidly converts DHAP into GAP
(these isomers can inter-convert). GAP is the substrate needed for the next step
of glycolysis.
Step 6
The enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serves
two functions in this reaction. First, it dehydrogenates GAP by transferring one
of its hydrogen (H⁺) molecules to the oxidizing agent nicotinamide adenine
dinucleotide (NAD⁺) to form NADH + H⁺.
Next, GAPDH adds a phosphate from the cytosol to the oxidized GAP to form
1,3-bisphosphoglycerate (BPG). Both molecules of GAP produced in the
previous step undergo this process of dehydrogenation and phosphorylation.
Step 7
The enzyme phosphoglycerokinase transfers a phosphate from BPG to a
molecule of ADP to form ATP. This happens to each molecule of BPG. This
reaction yields two 3-phosphoglycerate (3 PGA) molecules and two ATP
molecules.
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Step 8
The enzyme phosphoglyceromutase relocates the P of the two 3 PGA
molecules from the third to the second carbon to form two 2-phosphoglycerate
(2 PGA) molecules.
Step 9
The enzyme enolase removes a molecule of water from 2-phosphoglycerate to
form phosphoenolpyruvate (PEP). This happens for each molecule of 2 PGA
from Step 8.
Step 10
The enzyme pyruvate kinase transfers a P from PEP to ADP to form pyruvate
and ATP. This happens for each molecule of PEP. This reaction yields two
molecules of pyruvate and two ATP molecules.
Significance of the Glycolysis Pathway
1. Glycolysis is the only pathway that is taking place in all the cells of the
body.
2. Glycolysis is the only source of energy in erythrocytes.
3. In Strenuous exercise, when muscle tissues lack enough oxygen,
anaerobic glycolysis forms the major source of energy for muscles.
4. The Glycolytic pathway may be considered as the preliminary step before
complete oxidation.
5. The glycolytic pathway provides carbon skeletons for synthesis of non
essential amino acids as well as glycerol part of fat.
6. Most of the reactions of the glycolytic pathway are reversible, which are
also used for gluconeogenesis.
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Energy Yield in Aerobic Glycolysis
Step Enzyme Source No. of ATP
1 Hexokinase – -1
3 Phosphofructokinase – -1
6 Glyceraldehyde-3- phosphate
dehydrogenase
NADH (+3) x 2 = +6
7 Phosphoglycerate kinase ATP (+1) x 2 = +2
10 Pyruvate kinase ATP (+1) x 2 = +2
Net
Yield
8 ATPs
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Krebs (Citric Acid) Cycle
It is also known as Tri Carboxylic Acid (TCA) cycle. In prokaryotic cells, the
citric acid cycle occurs in the cytoplasm; in eukaryotic cells, the citric acid cycle
takes place in the matrix of the mitochondria.
The cycle was first elucidated by scientist “Sir Hans Adolf Krebs” (1900 to
1981). He shared the Nobel Prize for physiology and Medicine in 1953 with
Fritz Albert Lipmann, the father of ATP cycle.
Figure 2 Citric Acid Cycle
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The process oxidises glucose derivatives, fatty acids and amino acids to carbon
dioxide (CO2) through a series of enzyme controlled steps. The purpose of the
Krebs Cycle is to collect (eight) high-energy electrons from these fuels by
oxidising them, which are transported by activated carriers NADH and FADH2
to the electron transport chain. The Krebs Cycle is also the source for the
precursors of many other molecules, and is therefore an amphibolic pathway
(meaning it is both anabolic and catabolic).
acetyl CoA + 3 NAD + FAD + ADP + HPO4-2 —————> 2 CO2 + CoA + 3
NADH+ + FADH+ + ATP
Reaction 1: Formation of Citrate
The first reaction of the cycle is the condensation of acetyl-
CoA with oxaloacetate to form citrate, catalyzed by citrate synthase.
Once oxaloacetate is joined with acetyl-CoA, a water molecule attacks the
acetyl leading to the release of coenzyme A from the complex.
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Reaction 2: Formation of Isocitrate
The citrate is rearranged to form an isomeric form, isocitrate by an
enzyme acontinase.
In this reaction, a water molecule is removed from the citric acid and then put
back on in another location. The overall effect of this conversion is that the –
OH group is moved from the 3′ to the 4′ position on the molecule. This
transformation yields the molecule isocitrate.
Reaction 3: Oxidation of Isocitrate to α-Ketoglutarate
In this step, isocitrate dehydrogenase catalyzes oxidative decarboxylation
of isocitrate to form α-ketoglutarate.
In the reaction, generation of NADH from NAD is seen. The enzyme isocitrate
dehydrogenase catalyzes the oxidation of the –OH group at the 4′ position of
isocitrate to yield an intermediate which then has a carbon dioxide molecule
removed from it to yield alpha-ketoglutarate.
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Reaction 4: Oxidation of α-Ketoglutarate to Succinyl-CoA
Alpha-ketoglutarate is oxidized, carbon dioxide is removed, and coenzyme A
is added to form the 4-carbon compound succinyl-CoA.
During this oxidation, NAD+ is reduced to NADH + H+. The enzyme that
catalyzes this reaction is alpha-ketoglutarate dehydrogenase.
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Reaction 5: Conversion of Succinyl-CoA to Succinate
CoA is removed from succinyl-CoA to produce succinate.
The energy released is used to make guanosine triphosphate (GTP) from
guanosine diphosphate (GDP) and Pi by substrate-level phosphorylation. GTP
can then be used to make ATP. The enzyme succinyl-CoA synthase catalyzes
this reaction of the citric acid cycle.
Reaction 6: Oxidation of Succinate to Fumarate
Succinate is oxidized to fumarate.
During this oxidation, FAD is reduced to FADH2. The enzyme succinate
dehydrogenase catalyzes the removal of two hydrogens from succinate.
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Reaction 7: Hydration of Fumarate to Malate
The reversible hydration of fumarate to L-malate is catalyzed by fumarase
(fumarate hydratase).
Fumarase continues the rearrangement process by adding Hydrogen and
Oxygen back into the substrate that had been previously removed.
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Reaction 8: Oxidation of Malate to Oxaloacetate
Malate is oxidized to produce oxaloacetate, the starting compound of the citric
acid cycle by malate dehydrogenase. During this oxidation, NAD+ is reduced
to NADH + H+.
ATP Generation
Total ATP = 12 ATP
3 NAD+ = 9 ATP
1 FAD = 2 ATP
1 ATP = 1 ATP
Reviewing the whole process, the Krebs cycle primarily transforms the acetyl
group and water, into carbon dioxide and energized forms of the other reactants.
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Significance of Krebs Cycle
1. Intermediate compounds formed during Krebs cycle are used for the
synthesis of biomolecules like amino acids, nucleotides, chlorophyll,
cytochromes and fats etc.
2. Intermediate like succinyl CoA takes part in the formation of chlorophyll.
3. Amino Acids are formed from α- Ketoglutaric acid, pyruvic acids and
oxaloacetic acid.
4. Krebs cycle (citric Acid cycle) releases plenty of energy (ATP) required
for various metabolic activities of cell.
By this cycle, carbon skeleton are got, which are used in process of growth and
for maintaining the cells.
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Pentose phosphate pathway (PPP) or Hexose mono-phosphate
(HMP) shunt
The hexose monophosphate shunt, also known as the pentose phosphate
pathway, is a unique pathway used to create products essential in the body for
many reasons. The HMP shunt is an alternative pathway to glycolysis and is
used to produce ribose-5-phosphate and nicotinamide adenine dinucleotide
phosphate (NADPH). This pathway occurs in the oxidative and non-oxidative
phases, each comprising a series of reactions. The HMP shunt also has
significance in the medical world, as enzyme or co-factor deficiencies can have
potentially fatal implications on the affected patients.
• Pentose phosphate pathway is an alternative pathway to glycolysis and
TCA cycle for oxidation of glucose.
• It is a shunt of glycolysis
• It is also known as hexose monophosphate (HMP) shunt or
phosphogluconate pathway.
• It occurs in cytoplasm of both prokaryotes and eukaryotes
• Pentose phosphate pathway starts with glucose and it is a multi-steps
reaction.
The sequence of reactions are divided into two types.
I) oxidative reaction phase
II) Non-oxidative reaction phase
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Figure 3 HMP Shunt
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Oxidative phase
Figure 4 Oxidative Phase
First four reactions are irreversible and oxidative in which glucose molecule is
oxidized twice generating two molecules of NADPH and glucose is converted
into Ribose-5 phosphate.
1st step: conversion of glucose to glucose-6 phosphate.
This reaction is catalyzed by the enzyme hexokinase and a molecule of
ATP is utilized. This reaction is actually a primary step of glycolysis.
2nd step: conversion of glucose-6 phosphate to 6-phosphogluconolactone.
This reaction is catalyzed by an enzyme glucose-6 phosphate
dehydrogenase (G6PD) in the presence of Mg++ ion.
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In this reaction a molecule of NADPH is produced.
3rd step: conversion of 6-phosphogluconolactone to 6-phosphogluconate
This reaction is a hydrolysis reaction catalyzed by hydrolase enzyme
4th step: conversion of 6-phosphogluconate to ribose-5 phosphate
This reaction is catalyzed by the enzyme 6-phosphogluconate
dehydrogenase to produce 3-keto-6-phosphogluconate which undergoes
decarboxylation to produce ribulose-5 phosphate.
In this reaction a molecule of NADPH is generated.
Non oxidative phase
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Oxidative reactions are followed by a series reversible sugar phosphate
inter-conversion reaction.
Ribulose-5-phosphate is epimerized to produce xylulose 5-phosphate in
the presence of enzyme phosphor pentose epimerase. Similarly ribulose-
5-phosphate is also keto-isomerized into ribose 5-phosphate.
Xylulose-5-phsphate transfer two carbon moiety to ribose 5-phospahate
in the presence of enzyme transketolase to form sedoheptulose-7-
phosphate and glyceraldehyde 3—phosphate.
Sedoheptulose -7-phosphate transfer three carbon moiety to
glyceraldehyde -3-phosphate to form fructose 6-
phopsphate and erythrose 4-phosphate in the presence of enzyme
transaldolase.
Transketolase enzyme catalyse the transfer of two carbon moiety from
Xylulose-5-phsphate to erythrose-4- phosphate to form fructose-6-
phosphate and glyceraldehyde-3-phosphate.
Fructose-6-phosphate and glyceraldehyde-3-phosphate is later enter into
glycolysis and kreb’s cycle.
The rate and direction of reversible reaction depends upon the needs of
cell.
If cell needs only NADPH then fructose-phosphate and glyceraldehyde-3-
phosphate are converted back to glucose by reverse glycolysis, otherwise
converted to pyruvate and enter TCA cycle generating ATPs.
Significance of Pentose phosphate pathway
HMP is only the cytoplasmic pathway that generates NADPH
NADPH is produced in this pathway acts as reducing agent during
biosynthesis of various molecules eg. fattyacids.
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This pathway generates 3, 4, 5, 6 and 7 carbon compounds which are
precursors for biosynthesis of other molecules. Eg nucleotides are
synthesized from ribose-5-phsophate.
Pentose phosphate pathway is very essential for cell lacking
mitochondria (eg.RBCs) for generation of NADPH.
Triose, tetrose, pentose, hexose and heptose sugar are generated as
intermediate products in pentose phosphate pathway.
NADPH is also used to reduce (detoxify) Hydrogen peroxide in cell.
Resistance to malaria in some Africans are associated with deficiency of
glucose-6-phosphate dehydrogenase enzyme because malarial parasites
depend upon HMP shunt to reduce glutathione in RBCs.
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Glycogen Metabolism
Glycogen is a readily mobilized storage form of glucose. It is a very large,
branched polymer of glucose residue that can be broken down to yield glucose
molecules when energy is needed. Most of the glucose residues in glycogen are
linked by α-1,4-glycosidic bonds. Branches at about every tenth residue are
created by α-1,6-glycosidic bonds. Recall that α-glycosidic linkages form open
helical polymers, whereas β linkages produce nearly straight strands that form
structural fibrils, as in cellulose
There are 6 major steps are involved in the Glycogenolysis:
Step 1: Glucose Phosphorylation
Glucose is phosphorylated into Glucose-6-Phosphate, a reaction that is common
to the first reaction in the pathway of glycolysis from Glucose.
This reaction is catalyzed by Hexokinase in Muscle and Glucokinase in the
Liver.
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Step 2: Glc-6-P To Glc-1-P Conversion
Glucose-6-P is converted to Glc-1-Phosphate in a reaction catalyzed by the
enzyme “Phosphoglucomutase”.
Glucose-6-P + Enz-P <—> Glucose-1,6-bis Phosphate + Enz <—
> Glucose-1-Phosphate + Enzyme-P
Step 3: Attachment of UTP to Glc-1-P
Glucose-1-P reacts with Uridine triphosphate (UTP) to form the active
nucleotide Uridine diphosphate Glucose (UDP-Glc). The reaction is catalyzed
by the enzyme “UDPGlc Pyrophosphorylase”.
UTP+ Glucose-1-P <-> UDPGlc+ PPi
Step 4: Attachment of UDP-Glc to Glycogen Primer
A small fragment of pre-existing glycogen must act as a “Primer” (also called
GLYCOGENIN) to initiate glycogen synthesis. The Glycogenin can accept
glucose from UDP-Glc.
The hydroxyl group of the amino acid tyrosine of Glycogenin is the site at
which the initial glucose unit is attached. The enzyme Glycogen initiator
synthase transfers the first molecule of Glucose to Glycogenin. Then glycogenin
itself takes up a for glucose residues to form a fragment of primer which serves
as an acceptor for the rest of the glucose molecules.
Step 5: Glycogen Synthesis by Glycogen Synthase
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Glycogen synthase, the enzyme transfers the Glucose from UDP-Glc to the non-
reducing end of Glycogen to form alpha 1,4-linkages.
Glycogen synthase catalyzes the synthesis of a linear unbranched molecule with
alpha-1,4-glycosidic linkages.
Step 6: Glycogen Branches Formation
In this step, the formation of branches is brought about by the action of a
branching enzyme, namely branching enzyme (amylo-[1—>4]—>[1—>6]-
transglucosidase).
This enzyme transfers a small fragment of five to eight glucose residues from
the non-reducing end of the glycogen chain to another glucose residue where it
is linked by the alpha-1,6 bond.
It leads to the formation of a new non-reducing end, besides the existing one.
The glycogen chain will be elongated and branched.
The overall reaction of Glycogenesis,
(Glucose)n + Glucose +2 ATP -> (Glucose)n+1 +2 ADP + Pi
Two ATP molecules will utilize in this process. One is required for
the phosphorylation of Glucose and the other is needed for conversion of UDP
to UTP.
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GLYCOGEN STORAGE DISEASE
A glycogen storage disease (GSD, also glycogenosis and dextrinosis) is
a metabolic disorder caused by enzyme deficiencies affecting
either glycogen synthesis, glycogen breakdown or glycol sis (glucose
breakdown), typically in muscles and/or liver cells.
GSD has two classes of cause: genetic and acquired.
Genetic GSD is caused by any inborn error of metabolism (genetically
defective enzymes) involved in these processes. In livestock, acquired GSD is
caused by intoxication with the alkaloid castanospermine.
Glycogen storage disease type I (also known as GSDI or von Gierke disease) is
an inherited disorder caused by the buildup of a complex sugar
called glycogen in the body's cells. The accumulation of glycogen in certain
organs and tissues, especially the liver, kidneys, and small intestines, impairs
their ability to function normally.
Signs and symptoms of this condition typically appear around the age of 3 or 4
months, when babies start to sleep through the night and do not eat as frequently
as newborns. Affected infants may have low blood sugar (hypoglycemia),
which can lead to seizures. They can also have a buildup of lactic acid in the
body (lactic acidosis), high blood levels of a waste product called uric acid
(hyperuricemia), and excess amounts of fats in the blood (hyperlipidemia). As
they get older, children with GSDI have thin arms and legs and short stature. An
enlarged liver may give the appearance of a protruding abdomen. The kidneys
may also be enlarged. Affected individuals may also have diarrhea and deposits
of cholesterol in the skin (xanthomas).
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People with GSDI may experience delayed puberty. Beginning in young to mid-
adulthood, affected individuals may have thinning of the bones (osteoporosis), a
form of arthritis resulting from uric acid crystals in the joints (gout), kidney
disease, and high blood pressure in the blood vessels that supply the lungs
(pulmonary hypertension). Females with this condition may also have abnormal
development of the ovaries (polycystic ovaries). In affected teens and adults,
tumors called adenomas may form in the liver. Adenomas are usually
noncancerous (benign), but occasionally these tumors can become cancerous
(malignant).
Researchers have described two types of GSDI, which differ in their signs and
symptoms and genetic cause. These types are known as glycogen storage
disease type Ia (GSDIa) and glycogen storage disease type Ib (GSDIb). Two
other forms of GSDI have been described, and they were originally named types
Ic and Id. However, these types are now known to be variations of GSDIb; for
this reason, GSDIb is sometimes called GSD type I non-a.
Many people with GSDIb have a shortage of white blood cells (neutropenia),
which can make them prone to recurrent bacterial infections. Neutropenia is
usually apparent by age 1. Many affected individuals also have inflammation of
the intestinal walls (inflammatory bowel disease). People with GSDIb may have
oral problems including cavities, inflammation of the gums (gingivitis), chronic
gum (periodontal) disease, abnormal tooth development, and open sores (ulcers)
in the mouth. The neutropenia and oral problems are specific to people with
GSDIb and are typically not seen in people with GSDIa.
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Gluconeogenesis
Gluconeogenesis (GNG) is a metabolic pathway that results in the generation
of glucose from certain non-carbohydrate carbon substrates. From breakdown
of proteins, these substrates include glucogenic amino acids (although
not ketogenic amino acids); from breakdown of lipids (such as triglycerides),
they include glycerol, odd-chain fatty acids (although not even-chain fatty acids,
see below); and from other steps in metabolism they
include pyruvate and lactate. Although most gluconeogenesis occurs in the
liver, the relative contribution of gluconeogenesis by the kidney is increased in
diabetes and prolonged fasting.
Gluconeogenesis is one of several main mechanisms used by humans and many
other animals to maintain blood glucose levels, avoiding low levels
(hypoglycemia). Other means include the degradation
of glycogen (glycogenolysis) and fatty acid catabolism.
Gluconeogenesis is a ubiquitous process, present in plants, animals, fungi,
bacteria, and other microorganisms. In vertebrates, gluconeogenesis takes place
mainly in the liver and, to a lesser extent, in the cortex of the kidneys.
In ruminants, this tends to be a continuous process. In many other animals, the
process occurs during periods of fasting, starvation, low-carbohydrate diets, or
intense exercise. The process is highly endergonic until it is coupled to the
hydrolysis of ATP or GTP, effectively making the process exergonic. For
example, the pathway leading from pyruvate to glucose-6-phosphate requires 4
molecules of ATP and 2 molecules of GTP to proceed spontaneously.
Gluconeogenesis is often associated with ketosis.
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In ruminants, because dietary carbohydrates tend to be metabolized
by rumen organisms, gluconeogenesis occurs regardless of fasting, low-
carbohydrate diets, exercise, etc.
Pathway
Gluconeogenesis is a pathway consisting of a series of eleven enzyme-catalyzed
reactions. The pathway will begin in either the liver or kidney, in the
mitochondria or cytoplasm of those cells, this being dependent on the substrate
being used. Many of the reactions are the reverse of steps found in glycolysis.
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Gluconeogenesis begins in the mitochondria with the formation of oxaloacetate
by the carboxylation of pyruvate. This reaction also requires one molecule
of ATP, and is catalyzed by pyruvate carboxylase. This enzyme is stimulated by
high levels of acetyl-CoA (produced in β-oxidation in the liver) and inhibited by
high levels of ADP and glucose.
Oxaloacetate is reduced to malate using NADH, a step required for its
transportation out of the mitochondria.
Malate is oxidized to oxaloacetate using NAD+ in the cytosol, where the
remaining steps of gluconeogenesis take place.
Oxaloacetate is decarboxylated and then phosphorylated to
form phosphoenolpyruvate using the enzyme PEPCK. A molecule
of GTP is hydrolyzed to GDP during this reaction.
The next steps in the reaction are the same as reversed glycolysis.
However, fructose 1,6-bisphosphatase converts fructose 1,6-
bisphosphate to fructose 6-phosphate, using one water molecule and
releasing one phosphate (in glycolysis, phosphofructokinase 1 converts
F6P and ATP to F1,6BP and ADP). This is also the rate-limiting step of
gluconeogenesis.
Glucose-6-phosphate is formed from fructose 6-
phosphate by phosphoglucoisomerase (the reverse of step 2 in glycolysis).
Glucose-6-phosphate can be used in other metabolic pathways or
dephosphorylated to free glucose. Whereas free glucose can easily diffuse
in and out of the cell, the phosphorylated form (glucose-6-phosphate) is
locked in the cell, a mechanism by which intracellular glucose levels are
controlled by cells.
The final gluconeogenesis, the formation of glucose, occurs in
the lumen of the endoplasmic reticulum, where glucose-6-phosphate is
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hydrolyzed by glucose-6-phosphatase to produce glucose and release an
inorganic phosphate. Like two steps prior, this step is not a simple reversal
of glycolysis, in which hexokinase catalyzes the conversion of glucose and
ATP into G6P and ADP. Glucose is shuttled into the cytoplasm by glucose
transporters located in the endoplasmic reticulum's membrane.
Significance
The Enzyme Pyruvate carboxylase, a deficiency is seen as an inborn error
of metabolism, where mental retardation is manifested.
Its incidence is one in 25,000 births.
Pyruvate carboxylase gene is located in human chromosome No. 11.
In type II diabetes mellitus condition, the risen Gluconeogenesis is
responsible for the production of excessive Glucose after an overnight
fast. A continual supply of Glucose is necessary as a source of energy,
especially for the Nervous system and the Erythrocytes.
Gluconeogenesis mechanism is used to clear the products of the
metabolism of other tissues from the blood, eg: Lactate, produced by
Muscle and erythrocytes and Glycerol, which is continuously produced
by adipose tissue.
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HORMONAL REGULATION OF BLOOD GLUCOSE LEVEL AND
DIABETES MELLITUS
Blood glucose regulation involves maintaining blood glucose levels at constant
levels in the face of dynamic glucose intake and energy use by the body.
Glucose is key in the energy intake of humans. On average this target range is
60-100 mg/dL for an adult although people can be asymptomatic at much more
varied levels. In order to maintain this range there are two main hormones that
control blood glucose levels: insulin and glucagon. Insulin is released when
there are high amounts of glucose in the blood stream.
Glucagon is released when there are low levels of glucose in the blood stream.
There are other hormones that effect glucose regulation and are mainly
controlled by the sympathetic nervous system. Blood glucose regulation is very
important to the maintenance of the human body. The brain doesn’t have any
energy storage of its own and as a result needs a constant flow of glucose, using
about 120 grams of glucose daily or about 60% of total glucose used by the
body at resting state. Without proper blood glucose regulation the brain and
other organs could starve leading to death.
Hormonal regulation of blood glucose level
Cells of the body require nutrients in order to function, and these nutrients are
obtained through feeding. In order to manage nutrient intake, storing excess
intake and utilizing reserves when necessary, the body uses hormones to
moderate energy stores. Insulin is produced by the beta cells of the pancreas,
which are stimulated to release insulin as blood glucose levels rise (for example,
after a meal is consumed). Insulin lowers blood glucose levels by enhancing the
rate of glucose uptake and utilization by target cells, which use glucose for ATP
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production. It also stimulates the liver to convert glucose to glycogen, which is
then stored by cells for later use. Insulin also increases glucose transport into
certain cells, such as muscle cells and the liver. This results from an insulin-
mediated increase in the number of glucose transporter proteins in cell
membranes, which remove glucose from circulation by facilitated diffusion. As
insulin binds to its target cell via insulin receptors and signal transduction, it
triggers the cell to incorporate glucose transport proteins into its membrane.
This allows glucose to enter the cell, where it can be used as an energy source.
However, this does not occur in all cells: some cells, including those in the
kidneys and brain, can access glucose without the use of insulin. Insulin also
stimulates the conversion of glucose to fat in adipocytes and the synthesis of
proteins. These actions mediated by insulin cause blood glucose concentrations
to fall, called a hypoglycemic “low sugar” effect, which inhibits further insulin
release from beta cells through a negative f Impaired insulin function can lead
to a condition called diabetes mellitus, the main symptoms of which are
illustrated in Figure 1. This can be caused by low levels of insulin production by
the beta cells of the pancreas, or by reduced sensitivity of tissue cells to insulin.
This prevents glucose from being absorbed by cells, causing high levels of
blood glucose, or hyperglycemia (high sugar). High blood glucose levels make
it difficult for the kidneys to recover all the glucose from nascent urine,
resulting in glucose being lost in urine. High glucose levels also result in less
water being reabsorbed by the kidneys, causing high amounts of urine to be
produced; this may result in dehydration. Over time, high blood glucose levels
can cause nerve damage to the eyes and peripheral body tissues, as well as
damage to the kidneys and cardiovascular system. Over secretion of insulin can
cause hypoglycemia, low blood glucose levels. This causes insufficient glucose
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availability to cells, often leading to muscle weakness, and can sometimes cause
unconsciousness or death if left untreated.
When blood glucose levels decline below normal levels, for example between
meals or when glucose is utilized rapidly during exercise, the
hormone glucagon is released from the alpha cells of the pancreas. Glucagon
raises blood glucose levels, eliciting what is called a hyperglycemic effect, by
stimulating the breakdown of glycogen to glucose in skeletal muscle cells and
liver cells in a process called glycogenolysis. Glucose can then be utilized as
energy by muscle cells and released into circulation by the liver cells. Glucagon
also stimulates absorption of amino acids from the blood by the liver, which
then converts them to glucose. This process of glucose synthesis is
called gluconeogenesis. Glucagon also stimulates adipose cells to release fatty
acids into the blood. These actions mediated by glucagon result in an increase in
blood glucose levels to normal homeostatic levels. Rising blood glucose levels
inhibit further glucagon release by the pancreas via a negative feedback
mechanism. In this way, insulin and glucagon work together to maintain
homeostatic glucose levels, as shown in Figure 2.
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Regulation of Blood Glucose Levels by Thyroid Hormones
The basal metabolic rate, which is the amount of calories required by the body
at rest, is determined by two hormones produced by the thyroid
gland: thyroxine, also known as tetraiodothyronine or T4,
and triiodothyronine, also known as T3. These hormones affect nearly every
cell in the body except for the adult brain, uterus, testes, blood cells, and spleen.
They are transported across the plasma membrane of target cells and bind to
receptors on the mitochondria resulting in increased ATP production. In the
nucleus, T3 and T4activate genes involved in energy production and glucose
oxidation. This results in increased rates of metabolism and body heat
production, which is known as the hormone’s calorigenic effect.
T3 and T4 release from the thyroid gland is stimulated by thyroid-stimulating
hormone (TSH), which is produced by the anterior pituitary. TSH binding at
the receptors of the follicle of the thyroid triggers the production of T3 and
T4 from a glycoprotein called thyroglobulin. Thyroglobulin is present in the
follicles of the thyroid, and is converted into thyroid hormones with the addition
of iodine. Iodine is formed from iodide ions that are actively transported into the
thyroid follicle from the bloodstream. A peroxidase enzyme then attaches the
iodine to the tyrosine amino acid found in thyroglobulin. T3 has three iodine
ions attached, while T4 has four iodine ions attached. T3 and T4 are then released
into the bloodstream, with T4 being released in much greater amounts than T3.
As T3is more active than T4 and is responsible for most of the effects of thyroid
hormones, tissues of the body convert T4 to T3 by the removal of an iodine ion.
Most of the released T3 and T4 becomes attached to transport proteins in the
bloodstream and is unable to cross the plasma membrane of cells. These
protein-bound molecules are only released when blood levels of the unattached
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hormone begin to decline. In this way, a week’s worth of reserve hormone is
maintained in the blood. Increased T3 and T4 levels in the blood inhibit the
release of TSH, which results in lower T3 and T4 release from the thyroid.
The follicular cells of the thyroid require iodides (anions of iodine) in order to
synthesize T3 and T4. Iodides obtained from the diet are actively transported into
follicle cells resulting in a concentration that is approximately 30 times higher
than in blood. The typical diet in North America provides more iodine than
required due to the addition of iodide to table salt. Inadequate iodine intake,
which occurs in many developing countries, results in an inability to synthesize
T3 and T4 hormones. The thyroid gland enlarges in a condition called goiter,
which is caused by overproduction of TSH without the formation of thyroid
hormone. Thyroglobulin is contained in a fluid called colloid, and TSH
stimulation results in higher levels of colloid accumulation in the thyroid. In the
absence of iodine, this is not converted to thyroid hormone, and colloid begins
to accumulate more and more in the thyroid gland, leading to goiter.
Disorders can arise from both the underproduction and overproduction of
thyroid hormones. Hypothyroidism, underproduction of the thyroid hormones,
can cause a low metabolic rate leading to weight gain, sensitivity to cold, and
reduced mental activity, among other symptoms. In children, hypothyroidism
can cause cretinism, which can lead to mental retardation and growth
defects. Hyperthyroidism, the overproduction of thyroid hormones, can lead to
an increased metabolic rate and its effects: weight loss, excess heat production,
sweating, and an increased heart rate. Graves’ disease is one example of a
hyperthyroid condition.
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UNIT- II
Biological oxidation-reduction reactions
Oxidation-reduction (or "redox") reactions are a very large class of chemical
reactions in which both oxidation and reduction necessarily occur.
An oxidation is defined as loss of electrons in the course of a chemical reaction.
If a species gains electrons, it is undergoing a reduction. Since electrons are
"conserved" in a chemical reaction (they are not created or destroyed), one
chemical species' loss is another's gain. Thus, a reduction cannot occur with a
corresponding oxidation, and vice-versa. The term "redox" also nicely
encapsulates how inextricably tied together oxidation and reduction are in
reality.
Other terminology used in discussing redox chemistry: A chemical species that
gets reduced is acting as an oxidizing agent, or oxidant, while the species
undergoing oxidation is acting as the reducing agent, or reductant.
Oxidation state (or oxidation number) is a bookkeeping device employed by
chemists to help them classify and understand chemical reactions. The simplest
way to interpret oxidation number is to think of it as the number of electrons
lost or gained by an atom (compared to its neutral, uncombined form) when it
reacts to form ions or molecules. Consider first the case of ions. For monatomic
ions, such as Na+ or Cl−, the oxidation number is the same as the charge, +1 and
−1, for the sodium cation and chloride anion, respectively. In molecules and
polyatomic ions, oxidation states for atoms are calculated by comparing the
number of valence electrons in the neutral atom with a count of the surrounding
bonding and nonbonding electrons in the Lewis structure. In this respect, it is
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similar to determining formal charge of an atom in a Lewis structure. A general
rule in determining an oxidation number of an atom in a complete Lewis
structure, is any differences in electronegativity between covalently bonded
atoms is treated as if the bond is actually ionic. That means both electrons are
counted as belonging to the more electronegative atom.
In computing formal charge, electrons in covalent bonds are treated as equally
shared, despite differences in electro negativity. But like formal charge, the sum
of the oxidation numbers for each atom in a formula or Lewis structure for a
molecular or ionic species must sum to the net charge of that formula or Lewis
structure (zero for a molecule). We will soon see that assignment of oxidation
numbers and following how they change in a chemical reaction allows us to
recognize redox reactions and determine the stoichiometry of the electron
transfer occurring.
It is easy to recognize any reaction featuring an uncombined, neutral element as
a redox reaction. Some examples are
CH4(g) + 2 O2(g) → CO2(g) + 2 H2O(l)
Fe(s) + O2(g) → Fe2O3(s)
2 Na(s) + Cl2(g) → 2 NaCl (s)
Identifying oxidation-reduction reactions using oxidation states
Applying these rules to the two previous reactions shows that the oxidation
states of O and H do not change from their usual values (−2, +1, respectively) in
either case. In the first reaction, tin is oxidized (its oxidation state is +2 on the
reactant side, and +4 on the product side, by rule 2), while the oxidation state of
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N in the nitrate anion is +5, and in nitrogen monoxide it is +2 (by rules 3 and 4),
so nitrogen undergoes reduction. In the second reaction, chromium is reduced -
its oxidation state is +6 in the dichromate (Cr2O72−) anion (by rules 3 and 4) on
the reactant side, and is +3 (by rule 2) on the product side. The two carbon
atoms in ethanol can be assigned an oxidation state of −2 (it is actually an
average in this case - two atoms of the same element can have different
oxidation states depending on how they are bonded in the molecule or ion under
consideration, but that need not concern us further here), while in the product
acetic acid, we arrive at an oxidation state of zero for carbon. We see that the
organic compound ethanol is being oxidized to acetic acid by the oxidizing
agent dichromate.
Oxidation states of carbon atoms in simple organic compounds
The oxidation levels of carbon atoms in various functional groups will be
considered in order to train us to recognize oxidation and reduction in
biochemical reactions. The following shows how the oxidation number of the
carbon atom changes for the series of one-carbon molecules containing C, H,
and O only.
Note the oxidation number for carbon changes in steps of two in concert with
the addition or loss of two electrons and as the number of bonds the carbon
atom makes to oxygen decreases or increases.
Having assigned oxidation numbers, and understanding that changes in
oxidation numbers are the result of adding electrons (reduction) or removing
electrons (oxidation), balanced half reactions for oxidations or reductions can be
written. For example, the reduction of formaldehyde to methanol shown above
can first be written as an unbalanced reduction half reaction,
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CH2O + 2e− → CH3OH
Since the equation is unbalanced with respect to charge and hydrogen atoms,
acidic conditions are typically assumed in cases such as this (meaning that we
add H+ on either side of the equation as required to balance both hydrogen
atoms and charge), and the balanced reduction half reaction can be written:
CH2O + 2H+ + 2e− → CH3OH
Reactive oxygen species
A similar series for the simplest oxygen compounds is of biological importance.
The energy-yielding process of oxidative phosphorylation results in the
reduction of molecular oxygen to water. Both oxygen and water are themselves
benign, such is not true for intermediates that are only partially reduced to
water. Although the molecular choreography of oxygen reduction in cells that
carry out oxidative metabolism is tightly and precisely channeled so as to
minimize the possibility of their release, occasionally and inevitably a partially
reduced oxygen species is produced.
Redox chemistry and electricity
Charge (q) is measured in Coulombs (C)
Charge of an electron: 1.60217653 × 10−19 C
The Faraday (F), the charge of 1 mol elementary charge:
F = 9.648534 × 104 C · mol−1 = 96.485 kJ ·V−1· mol−1
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Current (I) is measured in amperes (A). An ampere is defined as 1 A = 1 C ·
s −1
Voltage and electrical work. The flow of water is a useful analogy for electric
current. The flow as measured by volume of water per unit time would be like
the flow of charge per unit time. Furthermore, the gravitational potential
difference (in height of a column or reservoir of water above the level where
flow is measured) is analogous to the electric potential difference existing
between two charge "reservoirs". When charge flows "downhill" through an
electric potential gradient, it can do useful work, just as a mass of water flowing
downhill toward lower gravitational potential can do the work of turning a
turbine (and thereby generating ....electric potential energy!). Electric potential
is measured in volts, where a volt is defined as 1 V = 1 J · C −1 . In other words,
a charge of 1 C moving through a potential difference of 1 V is equivalent to 1 J
of work (work is equivalent to energy, as shown by the work-energy theorem of
physics). Note that 1 J · V −1 = 1 C is a convenient conversion factor for charge.
(This is used above in the unit conversion for the Faraday constant.) For reasons
of clarity, the potential difference quantity, also referred to as electromotive
force (emf), will be represented here as ΔE.
Redox processes and electron carrier molecules in biochemistry
A great many biochemical reactions are oxidation-reduction reactions, so in a
sense the participants in these reactions that undergo oxidation or reduction are
electron carriers. In order for redox processes to serve as a source of energy in
the form of an emf through which electrons flow, organisms utilize
(heterotrophs) or are able to create (autotrophs) molecules that are reducing
agents. These electrons get passed from one electron carrier to the next, in a
series of electron transfer reactions. Electrons flow exergonically from species
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with a lower to a higher reduction potential. Reduction of a terminal electron
acceptor, such as O2 in aerobic metabolism yields an end product which is
exchanged with surroundings.
There are recurring patterns of redox reactions in biochemistry. Metabolic
intermediates that are relatively reduced, derived for example from the the food
consumed by heterotrophic organisms, such as glucose 6-phosphate or pyruvate,
can be oxidized with electrons being transferred to the oxidized forms of redox
cofactors that serve as modular cosubstrates in enzyme-catalyzed redox
reactions. The enzymes involved are in the oxidoreductase class of enzymes,
and many of these work with one of two modular cosubstrates, nicotinamide
adenine dinucleotide and flavin adenine dinucleotide.
Nicotinamide adenine dinucleotide (NAD) is an important, ubiquitous redox
cofactor that functions as a carrier of electron pairs. The oxidized form of the
cofactor carries a positive charge, and is denoted NAD+ while the reduced form
is NADH. The nicotinamide portion of NAD+, consisting of a carbamylated
pyridine ring (in red in the figure below, corresponding to niacin, one of the B-
complex vitamins acts as the electron pair acceptor. In undergoing reduction,
the 4-position of the NAD+ ring (para to the nitrogen atom) in effect accepts a
hydride ion (H:−). Note that the "dinucleotide" part of the name is due to the fact
that the other "half" of NAD is an adenine-containing nucleotide, AMP. The
two "halves" of the molecule are linked by a phosphoanhydride bond.
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A closely related cofactor, NADP+, (reduced form, NADPH) differs only in
having a phosphate attached to the 2′ position of the ribose attached to adenine.
NADPH generally functions in redox reactions in biosynthetic pathways (e.g.
fatty acid synthesis, whereas NAD+ predominates in catabolic processes, such
as those associated with glycolysis and oxidative phosphorylation.
The NAD+/NADH redox half-reaction (see figure below) has a standard
biochemical reduction potential, ΔE°′ of −0.315 V. The progress of reactions
involving NAD+/NADH can be conveniently monitored spectrophotometrically
due to the appearance of a broad absorption with its peak at 340 nm
when NADH is formed.
The major source of NADH in oxidative metabolism is the citric acid
cycle. The NADH produced is reoxidized to NAD+ when the former donates its
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electrons to the first component of the electron transport chain (ETC).
Eventually these electrons reduce molecular oxygen to water.
Flavin adenine dinucleotide (FAD) is an important, ubiquitous redox cofactor,
consisting of (like NAD) an AMP moiety in an anhydride linkage
to FMN (flavin mononucleotide, shown in blue in the figure).
The isoalloxazine ring system of FAD or FMN can accept one or two electrons,
in contrast to NAD, which can only be reduced by two electrons at a time. In
the electron transport chain (ETC) FMN acts as a cofactor in the NADH-Q
oxidoreductase complex, accepting 2 electrons from NADH, and transferring
them to a series of iron-sulfur (Fe-S) proteins, and then to coenzyme Q
(ubiquinone). Electrons from FADH2 enter the electron transport chain at the
level of cytochrome reductase, the second proton-pumping complex in the
electron transport chain (downstream from NADH-Q reductase), via
the succinate-Q oxidoreductase complex, which accomplishes the transfer of
electrons from FADH2 to coenzyme Q (again through Fe-S proteins). The full
structure of FAD is shown below. The three rings at the top constitute the
isoalloxazine ring system, or flavin portion of the molecule. The portion of the
molecule corresponding to FMN (shown in blue) also includes the residue of the
five-carbon D-ribitol (a polyhydroxy alcohol derived from the sugar D-
ribose with an attached phosphate group. The isoalloxazine ring plus ribitol
corresponds to riboflavin, one of the B-complex vitamins.
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FMN is linked to an adenosine monophosphate (AMP) shown in black) by a
phosphodiester bond. The reduction of FAD involves the 1 and 5
nitrogen atoms (labelled red in the figure at left), and the oxidation states of
FAD/FMN are shown in the figure below. The molecule labelled (1) represents
FAD or FMN - the most oxidized form. The molecule labelled (2) is a radical
or semiquinone formed by a one electron reduction of (1). A second one-
electron reduction converts the radical to (3), which represents the fully reduced
forms FADH2 or FMH2.
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Electron Transport Chain
Definition
The electron transport chain is a cluster of proteins that transfer electrons
through a membrane within mitochondria to form a gradient of protons that
drives the creation of adenosine triphosphate (ATP). ATP is used by the cell as
the energy for metabolic processes for cellular functions.
During the process, a proton gradient is created when the protons are pumped
from the mitochondrial matrix into the intermembrane space of the cell, which
also helps in driving ATP production. Often, the use of a proton gradient is
referred to as the chemiosmotic mechanism that drives ATP synthesis since it
relies on a higher concentration of protons to generate “proton motive force”.
The amount of ATP created is directly proportional to the number of protons
that are pumped across the inner mitochondrial membrane.
The electron transport chain involves a series of redox reactions that relies on
protein complexes to transfer electrons from a donor molecule to an acceptor
molecule. As a result of these reactions, the proton gradient is produced,
enabling mechanical work to be converted into chemical energy, allowing ATP
synthesis. The complexes are embedded in the inner mitochondrial membrane
called the cristae in eukaryotes. Enclosed by the inner mitochondrial membrane
is the matrix, which is where necessary enzymes such
as pyruvate dehydrogenase and pyruvate carboxylase are located. The process
can also be found in photosynthetic eukaryotes in the thylakoid membrane of
chloroplasts and in prokaryotes, but with modifications.
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By-products from other cycles and processes, like the citric acid cycle, amino
acid oxidation, and fatty acid oxidation, are used in the electron transport chain.
As seen in the overall redox reaction,
2 H+ + 2 E+ + ½ O2 → H2O + ENERGY
energy is released in an exothermic reaction when electrons are passed through
the complexes; three molecules of ATP are created. Phosphate located in the
matrix is imported via the proton gradient, which is used to create more ATP.
The process of generating more ATP via the phosphorylation of ADP is referred
to oxidative phosphorylation since the energy of hydrogen oxygenation is used
throughout the electron transport chain. The ATP generated from this reaction
go on to power most cellular reactions necessary for life.
Mechanism of ETC
In the electron transfer chain, electrons move along a series of proteins to
generate an expulsion type force to move hydrogen ions, or protons, across the
mitochondrial membrane. The electrons begin their reactions in Complex I,
continuing onto Complex II, traversed to Complex III and cytochrome c
via coenzyme Q, and then finally to Complex IV. The complexes themselves
are complex-structured proteins embedded in the phospholipid membrane. They
are combined with a metal ion, such as iron, to help with proton expulsion into
the intermembrane space as well as other functions. The complexes also
undergo conformational changes to allow openings for the transmembrane
movement of protons.
These four complexes actively transfer electrons from an organic metabolite,
such as glucose. When the metabolite breaks down, two electrons and a
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hydrogen ion are released and then picked up by the coenzyme NAD+ to
become NADH, releasing a hydrogen ion into the cytosol.
Fig.1 Electron transport chain
The NADH now has two electrons passing them onto a more mobile molecule,
ubiquinone (Q), in the first protein complex (Complex I). Complex I, also
known as NADH dehydrogenase, pumps four hydrogen ions from the matrix
into the intermembrane space, establishing the proton gradient. In the next
protein, Complex II or succinate dehydrogenase, another electron carrier and
coenzyme, succinate is oxidized into fumarate, causing FAD (flavin-adenine
dinucleotide) to be reduced to FADH2. The transport molecule, FADH2 is then
reoxidized, donating electrons to Q (becoming QH2), while releasing another
hydrogen ion into the cytosol. While Complex II does not directly contribute to
the proton gradient, it serves as another source for electrons.
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Fig.2 Electron transport chain in cell membrane
Complex III, or cytochrome c reductase, is where the Q cycle takes place. There
is an interaction between Q and cytochromes, which are molecules composed of
iron, to continue the transfer of electrons. During the Q cycle, the ubiquinol
(QH2) previously produced donates electrons to ISP and cytochrome b
becoming ubiquinone. ISP and cytochrome b are proteins that are located in the
matrix that then transfers the electron it received from ubiquinol to cytochrome
c1. Cytochrome c1 then transfers it to cytochrome c, which moves the electrons
to the last complex. (Note: Unlike ubiquinone (Q), cytochrome c can only carry
one electron at a time). Ubiquinone then gets reduced again to QH2, restarting
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the cycle. In the process, another hydrogen ion is released into the cytosol to
further create the proton gradient.
The cytochromes then extend into Complex IV, or cytochrome c oxidase.
Electrons are transferred one at a time into the complex from cytochrome c. The
electrons, in addition to hydrogen and oxygen, then react to form water in an
irreversible reaction. This is the last complex that translocates four protons
across the membrane to create the proton gradient that develops ATP at the end.
As the proton gradient is established, F1F0 ATP synthase, sometimes referred to
as Complex V, generates the ATP. The complex is composed of several
subunits that bind to the protons released in prior reactions. As the protein
rotates, protons are brought back into the mitochondrial matrix, allowing ADP
to bind to free phosphate to produce ATP. For every full turn of the protein,
three ATP is produced, concluding the electron transport chain.
Oxidative phosphorylation
Definition
Oxidative phosphorylation (UK or electron transport-linked phosphorylation) is
the metabolic pathway in which cells use enzymes to oxidize nutrients, thereby
releasing the chemical energy of molecular oxygen, which is used to produce
adenosine triphosphate (ATP). In most eukaryotes, this takes place inside
mitochondria. Almost all aerobic organisms carry out oxidative
phosphorylation. This pathway is so pervasive because the energy of the double
bond of oxygen is so much higher than the energy of the double bond in
carbondioxide or in pairs of single bonds in organic molecules observed in
alternative fermentation processes such as anaerobic glycolysis.
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Overview:
Fig. 3 Simple diagram of the electron transport chain
The electron transport chain is a series of proteins embedded in the inner
mitochondrial membrane.
In the matrix, NADH and FADH2 deposit their electrons in the chain (at the
first and second complexes of the chain, respectively).
The energetically "downhill" movement of electrons through the chain causes
pumping of protons into the intermembrane space by the first, third, and fourth
complexes.
Finally, the electrons are passed to oxygen, which accepts them along with
protons to form water.
The proton gradient produced by proton pumping during the electron transport
chain is used to synthesize ATP. Protons flow down their concentration gradient
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into the matrix through the membrane protein ATP synthase, causing it to spin
(like a water wheel) and catalyze conversion of ADP to ATP.
The electron transport chain is a series of proteins and organic molecules
found in the inner membrane of the mitochondria. Electrons are passed from
one member of the transport chain to another in a series of redox reactions.
Energy released in these reactions is captured as a proton gradient, which is then
used to make ATP in a process called chemiosmosis. Together, the electron
transport chain and chemiosmosis make up oxidative phosphorylation. The
key steps of this process, shown in simplified form in the diagram above,
include:
Delivery of electrons by NADH and FADH_22start subscript, 2, end
subscript. Reduced electron carriers (NADH and FADH_22start
subscript, 2, end subscript) from other steps of cellular respiration transfer
their electrons to molecules near the beginning of the transport chain. In
the process, they turn back into NAD^++start superscript, plus, end
superscript and FAD, which can be reused in other steps of cellular
respiration.
Electron transfer and proton pumping. As electrons are passed down
the chain, they move from a higher to a lower energy level, releasing
energy. Some of the energy is used to pump H^++start superscript, plus,
end superscript ions, moving them out of the matrix and into the
intermembrane space. This pumping establishes an electrochemical
gradient.
Splitting of oxygen to form water. At the end of the electron transport
chain, electrons are transferred to molecular oxygen, which splits in half
and takes up H^++start superscript, plus, end superscript to form water.
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Gradient-driven synthesis of ATP. As H^++start superscript, plus, end
superscript ions flow down their gradient and back into the matrix, they
pass through an enzyme called ATP synthase, which harnesses the flow
of protons to synthesize ATP.
We'll look more closely at both the electron transport chain and chemiosmosis
in the sections below.
Substrate-level phosphorylation
Substrate-level phosphorylation is a metabolic reaction that results in the
formation of ATP or GTP by conversion of a higher energy substrate (whether
phosphate group attached or not) into lower energy product and a using some of
the released chemical energy, the Gibbs free energy, to transfer
a phosphoryl (PO3) group to ADP or GDP from another phosphorylated
compound.
Unlike oxidative phosphorylation, oxidation and phosphorylation are not
coupled in the process of substrate-level phosphorylation, and reactive
intermediates are most often gained in the course of oxidation processes
in catabolism. Most ATP is generated by oxidative phosphorylation in aerobic
or anaerobic respiration while substrate-level phosphorylation provides a
quicker, less efficient source of ATP, independent of external electron
acceptors. This is the case in human erythrocytes, which have no mitochondria,
and in oxygen-depleted muscle.
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Adenosine triphosphate is a major "energy currency" of the cell. The high
energy bonds between the phosphate groups can be broken the power a variety
of reactions used in all aspects of cell function.
Substrate-level phosphorylation occurs in the cytoplasm of cells
during glycolysis and in mitochondria either during the Krebs cycle or
by MTHFD1L , an enzyme interconverting ADP + phosphate + 10-
formyltetrahydrofolate to ATP + formate + tetrahydrofolate (reversibly), under
both aerobic and anaerobic conditions. In the pay-off phase of glycolysis, a net
of 2 ATP are produced by substrate-level phosphorylation.
Glycolysis
The first substrate-level phosphorylation occurs after the conversion of 3-
phosphoglyceraldehyde and Pi and NAD+ to 1,3-bisphosphoglycerate
via glyceraldehyde 3-phosphate dehydrogenase. 1,3-bisphosphoglycerate is then
dephosphorylated via phosphoglycerate kinase, producing 3-phosphoglycerate
and ATP through a substrate-level phosphorylation.
The second substrate-level phosphorylation occurs by
dephosphorylating phosphoenolpyruvate, catalyzed by pyruvate kinase,
producing pyruvate and ATP.
During the preparatory phase, each 6-carbon glucose molecule is broken into
two 3-carbon molecules. Thus, in glycolysis dephosphorylation results in the
production of 4 ATP. However, the prior preparatory phase consumes 2 ATP,
so the net yield in glycolysis is 2 ATP. 2 molecules of NADH are also produced
and can be used in oxidative phosphorylation to generate more ATP.
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Mitochondria
ATP can be generated by substrate-level phosphorylation in mitochondria in a
pathway that is independent from the proton motive force. In the matrix there
are three reactions capable of substrate-level phosphorylation, utilizing
either phosphoenolpyruvate carboxykinase or succinate-CoA ligase,
or monofunctional C1-tetrahydrofolate synthase.
Phosphoenolpyruvate carboxykinase
Mitochondrial phosphoenolpyruvate carboxykinase is thought to participate in
the transfer of the phosphorylation potential from the matrix to the cytosol and
vice versa. However, it is strongly favored towards GTP hydrolysis, thus it is
not really considered as an important source of intra-mitochondrial substrate-
level phosphorylation.
Succinate-CoA ligase
Succinate-CoA ligase is a heterodimer composed of an invariant α-subunit and a
substrate-specific ß-subunit, encoded by either SUCLA2 or SUCLG2. This
combination results in either an ADP-forming succinate-CoA ligase or a GDP-
forming succinate-CoA ligase. The ADP-forming succinate-CoA ligase is
potentially the only matrix enzyme generating ATP in the absence of a proton
motive force, capable of maintaining matrix ATP levels under energy-limited
conditions, such as transient hypoxia.
Monofunctional C1-tetrahydrofolate synthase
This enzyme is encoded by MTHFD1L and reversibly interconverts ADP +
phosphate + 10-formyltetrahydrofolate to ATP + formate + tetrahydrofolate.
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Other mechanisms
In working skeletal muscles and the brain, Phosphocreatine is stored as a readily
available high-energy phosphate supply, and the enzyme creatine
phosphokinase transfers a phosphate from phosphocreatine to ADP to produce
ATP. Then the ATP releases giving chemical energy. This is sometimes
erroneously considered to be substrate-level phosphorylation, although it is
a transphosphorylation.
Importance of substrate-level phosphorylation in anoxia
During anoxia, provision of ATP by substrate-level phosphorylation in the
matrix is important not only as a mere means of energy, but also to prevent
mitochondria from straining glycolytic ATP reserves by maintaining
the adenine nucleotide translocator in ‘forward mode’ carrying ATP towards the
cytosol.
Oxidative phosphorylation
An alternative method used to create ATP is through oxidative phosphorylation,
which takes place during cellular respiration. This process utilizes the oxidation
of NADH to NAD+, yielding 3 ATP, and of FADH2 to FAD, yielding 2 ATP.
The potential energy stored as an electrochemical gradient of protons (H+)
across the inner mitochondrial membrane is required to generate ATP from
ADP and Pi (inorganic phosphate molecule), a key difference from substrate-
level phosphorylation. This gradient is exploited by ATP synthase acting as a
pore, allowing H+ from the mitochondrial intermembrane space to move down
its electrochemical gradient into the matrix and coupling the release of free
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energy to ATP synthesis. Conversely, electron transfer provides the energy
required to actively pump H+ out of the matrix.
Uncouplers of oxidative phosphorylation
Uncouplers of oxidative phosphorylation in mitochondria inhibit the coupling
between the electron transport and phosphorylation reactions and thus inhibit
ATP synthesis without affecting the respiratory chain and ATP synthase.
Uncouplers inhibit ATP synthesis by preventing this coupling reaction in such a
fashion that the energy produced by redox reactions cannot be used for
phosphorylation. Uncouplers include DNP, valinomycin, and CCCP. Most of
them are hydrophobic weak acids that act by protonophoric action and
activities.
Uncouplers of oxidative phosphorylation in mitochondria inhibit the coupling
between the electron transport and phosphorylation reactions and thus inhibit
ATP synthesis without affecting the respiratory chain and ATP synthase (H(+)-
ATPase). Miscellaneous compounds are known to be uncouplers, but weakly
acidic uncouplers are representative because they show very potent activities.
The most potent uncouplers discovered so far are the hindered phenol SF 6847,
and hydrophobic salicylanilide S-13, which are active in vitro at concentrations
in the 10 nM range. For induction of uncoupling, an acid dissociable group,
bulky hydrophobic moiety and strong electron-withdrawing group are required.
Weakly acidic uncouplers are considered to produce uncoupling by their
protonophoric action in the H(+)-impermeable mitochondrial membrane. For
exerting these effects, the stability of the respective uncoupler anions in the
hydrophobic membrane is very important. High stability is achieved by
delocalization of the polar ionic charge through uncoupler (chemical)-specific
mechanisms. Such an action of weakly acidic uncouplers is characteristic of the
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highly efficient membrane targeting action of a nonsite-specific type of
bioactive compound.
One example of an ‘uncoupler’ of oxidative phosphorylation is DNP (2,4-
dinitrophenol).
2,4-Dinitrophenol (DNP), C6H4N2O5, is a cellular metabolic poison. It
uncouples oxidative phosphorylation by carrying protons across the
mitochondrial membrane, leading to a rapid consumption of energy without
generation of ATP.
In living cells, DNP acts as a proton ionophore, an agent that can shuttle protons
(hydrogen ions) across biological membranes. It defeats the proton gradient
across mitochondrial membrane, collapsing the proton motive force that the cell
uses to produce most of its ATP chemical energy. Instead of producing ATP,
the energy of the proton gradient is lost as heat.
DNP is often used in biochemistry research to help explore the bioenergetics of
chemiosmotic and other membrane transport processes.
The HMP shunt represents an alternative pathway for the breakdown of glucose.
Briefly describe the main products produced by this pathway and it’s biological
significance.
The main product are Ribose-5-P, NADPH and Intermediates of the glycolytic
pathway. HMP shunt represents an alternate degradative pathway for the
breakdown of glucose, and it provides a link between glycolysis and nucleotide
metabolism and fatty acid.
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Biological significance of Ribose-5-P is that serves as the precursor to various
nucleotides (ATP, NAD, NADP, coenzyme A) and nucleic acids (DNA) within
our cells.
Biological significance of NADPH: represents the major source of reducing
power for biosynthetic reactions within cells, particularly the synthesis of fatty
acids. It follows that the HMP shunt is active in tissues specialized for the
synthesis of fatty acids or steroids.
Biological significance of Intermediates of the glycolytic pathway: the demand
for NADPH in the cell is usually far greater than the demand for ribose-5-P,
thus the second phase of this pathway is devoted to recycling the 5-carbon
skeletons into intermediates of the glycolytic pathway so that the cell can
harness the energy that is present in these molecules.