Basic principles
of metabolism: catabolism, anabolism. Common pathways of proteins,
carbohydrates and lipids transformation.
Studying of Krebs cycle functioning
INVESTIGATION OF BIOLOGICAL
OXIDATION, OXIDATIVE PHOSPHORYLATION AND ATP SYNTHESIS. INHIBITORS AND UNCOUPLERS OF OXIDATIVE
PHOSPHORYLATION.
Metabolism is the set of life-sustaining chemical transformations within the cells of living organisms. These enzyme-catalyzed
reactions allow organisms to grow and reproduce, maintain their structures, and
respond to their environments. The word metabolism can also refer to all
chemical reactions that occur in living organisms, including digestion and the
transport of substances into and between different cells, in which case the set
of reactions within the cells is called intermediary metabolism or intermediate metabolism.
The term metabolism is derived from the Greek Μεταβολισμός –
"Metabolismos" for "change", or "overthrow". The history of the scientific study of
metabolism spans several centuries and has moved from examining whole animals
in early studies, to examining individual metabolic reactions in modern
biochemistry. The first controlled experiments in human metabolism were published by Santorio
Santorioin
In these early studies,
the mechanisms of these metabolic processes had not been identified and a vital force was thought to animate living tissue. In the 19th century, when studying the fermentation of
sugar to alcohol by yeast, Louis Pasteur concluded that fermentation was
catalyzed by substances within the yeast cells he called "ferments".
He wrote that "alcoholic fermentation is an act correlated with the life
and organization of the yeast cells, not with the death or putrefaction of the
cells."] This discovery, along with the publication
by Friedrich
Wöhler in 1828 of
the chemical synthesis of urea, notable for being the first organic compound
prepared from wholly inorganic precursors, proved that the organic compounds
and chemical reactions found in cells were no different in principle than any
other part of chemistry.
Metabolism is a term
that is used to describe all chemical reactions involved in maintaining the
living state of the cells and the organism. Metabolism can be conveniently
divided into two categories:
·
Catabolism - the breakdown of molecules
to obtain energy
·
Anabolism - the synthesis of all compounds
needed by the cells
Anabolism is the set of constructive metabolic processes where the energy released by
catabolism is used to synthesize complex molecules. In general, the complex
molecules that make up cellular structures are constructed step-by-step from
small and simple precursors. Anabolism involves three basic stages. Firstly,
the production of precursors such as amino acids, monosaccharides,isoprenoids and nucleotides,
secondly, their activation into reactive forms using energy from ATP, and
thirdly, the assembly of these precursors into complex molecules such as proteins, polysaccharides, lipids and nucleic acids.
Metabolism refers to
the highly integrated network of chemical reactions by which living cells grow
and sustain themselves. This network is composed of two major types of
pathways: anabolism and catabolism. Anabolism uses energy stored in the form of
adenosine triphosphate (ATP) to build larger molecules from smaller molecules.
Catabolic reactions degrade larger molecules in order to produce ATP and raw materials
for anabolic reactions.
Together, these two
general metabolic networks have three major functions:
(1) to extract energy from nutrients or solar
energy;
(2) to synthesize the
building blocks that make up the large molecules of life: proteins, fats,
carbohydrates, nucleic acids, and combinations of these substances;
(3) to synthesize and
degrade molecules required for special functions in the cell.
The series of products
created by the sequential enzymatic steps of anabolismor catabolism are called
metabolic intermediates, or metabolites. Each steprepresents a small change in
the molecule, usually the removal, transfer, oraddition of a specific atom,
molecule, or group of atoms that serves as a functional group, such as the
amino groups (-NH2) of proteins.
Most such metabolic
pathways are linear, that is, they begin with a specificsubstrate and end with
a specific product. However, some pathways, such as the Krebs cycle, are
cyclic. Often, metabolic pathways also have branches thatfeed into or out of
them. The specific sequences of intermediates in the pathways of cell
metabolism are called intermediary metabolism.
Among the many hundreds
of chemical reactions there are only a few that are central to the activity of
the cell, and these pathways are identical in mostforms of life.
Catabolic reactions are used to capture
and save energy from nutrients, as well as to degrade larger molecules into
smaller, molecular raw materials for reuse by the cell. The energy is stored in
the form of energy-rich ATP, whichpowers the reactions of anabolism. The useful
energy of ATP is stored in theform of a high-energy bond between the second and
third phosphate groups of ATP. The cell makes ATP by adding a phosphate group
to the molecule adenosinediphosphate (ADP). Therefore, ATP is the major
chemical link between the energy-yielding reactions of catabolism, and the
energy-requiring reactions of anabolism.
In some cases, energy is also conserved
as energy-rich hydrogen atoms in thecoenzyme nicotinamide adenine dinucleotide
phosphate in the reduced form of NADPH. The NADPH can then be used as a source
of high-energy hydrogen atoms during certain biosynthetic reactions of
anabolism.
In addition to the obvious difference in
the direction of their metabolic goals, anabolism and catabolism differ in
other significant ways. For example, the various degradative pathways of
catabolism are convergent. That is, many hundreds of different proteins,
polysaccharides, and lipids are broken down into relatively few catabolic end
products. The hundreds of anabolic pathways,however, are divergent. That is,
the cell uses relatively few biosynthetic precursor molecules to synthesize a
vast number of different proteins, polysaccharides, and lipids.
The opposing pathways of anabolism and
catabolism may also use different reaction intermediates or different enzymatic
reactions in some of the steps. Forexample, there are 11 enzymatic steps in the
breakdown of glucose into pyruvic acid in the liver. But the liver uses only
nine of those same steps in thesynthesis of glucose, replacing the other two
steps with a different set ofenzyme-catalyzed reactions. This occurs because the
pathway to degradation ofglucose releases energy, while the anabolic process of
glucose synthesis requires energy. The two different reactions of anabolism are
required to overcome the energy barrier that would otherwise prevent the
synthesis of glucose.
Another reason for having slightly
different pathways is that the corresponding anabolic and catabolic routes must
be independently regulated. Otherwise,if the two phases of metabolism shared
the exact pathway (only in reverse) aslowdown in the anabolic pathway would
slow catabolism, and vice versa.
Read more: http://www.faqs.org/health/topics/40/Metabolism.html#ixzz2QG2PLkrF
Some reactions can be
either catabolic or anabolic, depending on the circumstances. Such reactions
are called amphibolic reactions. Many of the reactions interconverting the
“simple molecules” fall in this category.
Catabolic and anabolic pathways are interrelated
in three ways:
Matter (catabolic pathways furnish the
precursor compounds for anabolism. Energy (catabolic pathways furnish the
energy to “drive” anabolism). Electrons (catabolic pathways furnish the
reducing power for anabolism).
Linear pathways convert one compound
through a series of intermediates to another compound. An example would be
glycolysis, where glucose is converted to pyruvate.
Branched pathways may either be
divergent (an intermediate can enter several linear pathways to different end
products) or convergent (several precursors can give rise to a common
intermediate). Biosynthesis of purines and of some amino acids are examples of
divergent pathways. There is usually some regulation at the branch point. The
conversion of various carbohydrates into the glycolytic pathway would be an
example of convergent pathways.
In a cyclic pathway, intermediates are
regenerated, and so some intermediates act in a catalytic fashion. In this
illustration, the cyclic pathway carries out the net conversion of X to Z. The
Tricarboxylic Acid Cycle is an example of a cyclic pathway.
A pool of compounds in equilibrium with
each other provides the intermediates for converting compounds to a variety of
products, depending on what is fed “into” the pool and what is “withdrawn” from
the pool. The phosphogluconate pathway is an example of such a pool of
intermediates. The pathway can convert glucose to CO2, hexoses to pentoses,
pentoses to hexoses, pentoses to trioses, etc. depending on what the cell
requires in a particular situation. NADPH as a source of reducing power for
anabolic reactions is also a main product of the phosphogluconate pathway.
Organisms differ in how
many of the molecules in their cells they can construct for themselves. Autotrophs such as plants can construct the
complex organic molecules in cells such as polysaccharides and proteins from
simple molecules like carbon
dioxide and water. Heterotrophs,
on the other hand, require a source of more complex substances, such as
monosaccharides and amino acids, to produce these complex molecules. Organisms
can be further classified by ultimate source of their energy: photoautotrophs
and photoheterotrophs obtain energy from light, whereas chemoautotrophs and
chemoheterotrophs obtain energy from inorganic oxidation reactions.
Metabolism is closely
linked to nutrition and the availability of nutrients. Bioenergetics is a term
which describes the biochemical or metabolic pathways by which the cell
ultimately obtains energy. Energy formation is one of the vital components of
metabolism.
Foods supply
carbohydrates in three forms: starch, sugar, and cellulose (fiber). Starches
and sugars form major and essential sources of energy for humans. Fibers
contribute to bulk in diet.
Body tissues depend on
glucose for all activities. Carbohydrates and sugars yield glucose by digestion or metabolism.Most people consume
around half of their diet as carbohydrates.
Proteins are the main
tissue builders in the body. They are part of every cell in the body. Proteins
help in cell structure, functions, haemoglobin formation to carry oxygen,
enzymes to carry out vital reactions and a myriad of other functions in the body.
Proteins are also vital in supplying nitrogen for DNA and RNA genetic material
and energy production.
Fats are concentrated
sources of energy. They produce twice as much energy as either carbohydrates or
protein on a weight basis.
Carbohydrate catabolism
is the breakdown of carbohydrates into smaller units. Carbohydrates are usually
taken into cells once they have been digested intomonosaccharides. Once inside, the major route of
breakdown is glycolysis,
where sugars such as glucose and fructose are converted into pyruvate and some ATP is generated.
Pyruvate is an intermediate in several
metabolic pathways, but the majority is converted to acetyl-CoA and fed into the citric acid
cycle. Although some more ATP is generated in the citric acid cycle,
the most important product is NADH, which is made from NAD+ as the acetyl-CoA is oxidized. This
oxidation releases carbon
dioxide as a waste
product. In anaerobic conditions, glycolysis produces lactate,
through the enzyme lactate dehydrogenase re-oxidizing NADH to NAD+ for re-use
in glycolysis. An alternative route for glucose breakdown is the pentose phosphate pathway, which reduces
the coenzyme NADPH and produces pentose sugars such asribose, the
sugar component of nucleic acids.
Fats are catabolised by hydrolysis to free fatty acids and glycerol. The
glycerol enters glycolysis and the fatty acids are broken down bybeta
oxidation to release
acetyl-CoA, which then is fed into the citric acid cycle. Fatty acids release
more energy upon oxidation than carbohydrates because carbohydrates contain
more oxygen in their structures.
Amino acids are either used to synthesize proteins
and other biomolecules, or oxidized to urea and carbon dioxide as a source of
energy. The oxidation pathway
starts with the removal of the amino group by a transaminase.
The amino group is fed into the urea cycle,
leaving a deaminated carbon skeleton in the form of a keto acid.
Several of these keto acids are intermediates in the citric acid cycle, for
example the deamination of glutamate forms α-ketoglutarate. The glucogenic amino acids can also be converted into glucose,
through gluconeogenesis .
Catabolism can be
broken down into 3 main stages.
Stage 1 – Stage
of Digestion
The large organic
molecules like proteins, lipids and polysaccharides are digested into
their smaller components outside cells. This stage acts on starch, cellulose or
proteins that cannot be directly absorbed by the cells and need to be broken
into their smaller units before they can be used in cell metabolism.
Digestive enzymes
include glycoside hydrolases that digest polysaccharides into monosaccharides or simple sugars.
The primary enzyme
involved in protein digestion is pepsin which catalyzes the nonspecific
hydrolysis of peptide bonds at an optimal pH of 2. In the lumen of the small
intestine, the pancreas secretes zymogens of trypsin,
chymotrypsin, elastase etc. These proteolytic enzymes break the proteins
down into free amino acids as well as dipeptides and tripeptides. The free
amino acids as well as the di and tripeptides are absorbed by the intestinal
mucosa cells which subsequently are released into the blood stream where they
are absorbed by other tissues.
The amino acids and
sugars are then pumped into cells by specific active transport proteins.
Stage 2 –
Release of energy
Once broken down these
molecules are taken up by cells and converted to yet smaller molecules, usually
acetyl coenzyme A (acetyl-CoA), which releases some energy.
Stage 3 - The acetyl group on the CoA is oxidised to water and carbon dioxide in
the citric acid cycle and electron transport chain, releasing the energy that
is stored by reducing the coenzyme nicotinamide adenine dinucleotide (NAD+)
into NADH.
When complex carbohydrates are broken they form simple sugars or
monosaccharides. This is taken up by the cells. Once inside these sugars
undergo glycolysis, where sugars such as glucose and fructose are converted
into pyruvate and some ATP is generated. Pyruvate is an intermediate in several
metabolic pathways, but the majority is converted to acetyl-CoA and fed into
the citric acid cycle or the Kreb’s cycle.
Within the citric acid
cycle more ATP is generated by the monosaccharides. The most important product
is NADH, which is made from NAD+ as the acetyl-CoA is oxidized. This
oxidation releases carbon dioxide as a waste product.
When there is no
oxygen, glycolysis produces lactate, through the enzyme lactate dehydrogenase,
re-oxidizing NADH to NAD+ for re-use in glycolysis.
Glucose can also be
broken down by pentose phosphate pathway, which reduces the coenzyme NADPH and
produces pentose sugars such as ribose, the sugar component of nucleic acids.
Proteins are broken
down into amino acids. Amino acids are either used to synthesize proteins and
other biomolecules, or oxidized to urea and carbon dioxide as a source of
energy.
In the process of
oxidation, first the amino group is removed by a transaminase. The amino group
is fed into the urea cycle, leaving a deaminated carbon skeleton in the form of
a keto acid.
These keto acids enter
the citric acid cycle. Glutamate, for example, forms α-ketoglutarate. Some
of the amines may also be converted into glucose, through gluconeogenesis.
Lipid breakdown
Fats are catabolised by
hydrolysis to free fatty acids and glycerol. The glycerol enters glycolysis and
the fatty acids are broken down by beta oxidation to release acetyl-CoA. This
acetyl co-A reaches the citric acid cycle next.
The chemical reactions
of metabolism are organized into metabolic pathways. These allow the basic
chemicals from nutrition to be transformed through a series of steps into
another chemical, by a sequence of enzymes.
Each metabolic pathway
consists of a series of biochemical reactions that are connected by their
intermediates: the products of one reaction are the substrates for subsequent reactions, and so on.
For example, one pathway may be responsible for the synthesis of a particular
amino acid, but the breakdown of that amino acid may occur via a separate and
distinct pathway. One example of an exception to this "rule" is the
metabolism of glucose. Glycolysis results in the breakdown of glucose,
but several reactions in the glycolysis pathway are reversible and participate
in the re-synthesis of glucose (gluconeogenesis).
·
·
Glycolysis was the first metabolic pathway
discovered:
1.
As glucose enters a cell, it is immediately phosphorylated by ATP to glucose 6-phosphate in the irreversible
first step.
2.
In times of excess lipid or protein energy sources, certain reactions in
the glycolysis pathway may run in reverse in order to
produce glucose 6-phosphate which is then used for storage as glycogen or starch.
·
Metabolic pathways are often regulated by feedback inhibition.
·
Some metabolic pathways flow in a
'cycle' wherein each component of the cycle is a substrate for the subsequent
reaction in the cycle, such as in the Krebs Cycle (see below).
·
Anabolic and catabolic pathways in eukaryotes often occur independently of each
other, separated either physically by compartmentalization within organelles or separated biochemically by the
requirement of different enzymes and co-factors.
Several distinct but
linked metabolic pathways are used by cells to transfer the energy released by
breakdown of fuel
molecules into ATPand other small molecules used for energy
(e.g. GTP, NADPH, FADH).
These pathways occur
within all living organisms in some form:
1.
Glycolysis
2.
Aerobic respiration and/or Anaerobic respiration
3.
Citric acid
cycle / Krebs cycle (not
in most obligate anaerobic organisms)
4.
Oxidative phosphorylation (not in obligate anaerobic organisms)
Catabolism
is characterized by convergence of three major routs toward a final common pathway.
Different proteins, fats and carbohydrates enter
the same pathway – tricarboxylic acid cycle.
Anabolism
can also be divided into stages, however the anabolic pathways are
characterized by divergence.
Monosaccharide
synthesis begin with CO2, oxaloacetate, pyruvate or lactate. Amino acids are synthesized from
acetyl CoA, pyruvate or keto acids of
Krebs cycle. .
Fatty acids
are constructed from acetyl CoA.
On the next stage monosaccharides, amino acids
and fatty acids are used for the synthesis of polysaccharides, proteins and fats.
Compartmentation of metabolic processes permits:
-
separate pools of metabolites within a cell
-
simultaneous operation of opposing metabolic paths
-
high local concentrations of metabolites
Example: fatty acid synthesis enzymes (cytosol), fatty acid breakdown enzymes (mitochondria).
Glycolysis
enzymes are located in the cytosol of cells. Pyruvate enters the mitochondrion
to be metabolized further.
Pyruvate dehydrogenase complex is a bridge
between glycolysis and aerobic metabolism – citric acid cycle.
Pyruvate freely diffuses
through the outer membrane of mitochon-dria through the channels formed by
transmembrane proteins porins.
Mitochondrial compartments:
The mitochondrial matrix contains
Pyruvate Dehydrogenase and enzymes of Krebs Cycle, plus other pathways such as
fatty acid oxidation.
The mitochondrial outer membrane
contains large channels, similar to bacterial porin channels, making the outer membrane leaky to ions and small
molecules.
The inner membrane is the major
permeability barrier of the mitochondrion. It contains various transport
catalysts, including a carrier protein that allows pyruvate to enter the
matrix. It is highly convoluted, with infoldings called cristae. Embedded in
the inner membrane are constituents of the respiratory chain and ATP Synthase.
Pyruvate Dehydrogenase
catalyzes oxidative decarboxylation of pyruvate, to form acetyl-CoA. The
overall reaction is shown below.
Pyruvate dehydrogenase complex is giant, with molecular mass ranging from 4 to 10 million
daltons.
Pyruvate Dehydrogenase is a large complex
containing many copies of each of three enzymes, E1, E2,
and E3.
The inner core of the mammalian Pyruvate
Dehydrogenase complex is an icosahedral structure consisting of 60
copies of E2.
At the periphery of the complex are:
·
30 copies of E1
(itself a tetramer with subunits a2b2) and
·
12 copies of E3
(a homodimer), plus 12 copies of an E3 binding protein
that links E3 to E2.
Prosthetic
groups are listed below
Enzyme |
Abbreviated |
Prosthetic Group |
Pyruvate Dehydrogenase |
E1 |
Thiamine pyrophosphate (TPP) |
Dihydrolipoyl Transacetylase |
E2 |
Lipoamide |
Dihydrolipoyl Dehydrogenase |
E3 |
FAD |
Thiamine pyrophosphate (TPP)
is a derivative of thiamine (vitamin B1). Nutritional
deficiency of thiamine leads to the disease beriberi. Beriberi
affects especially the brain, because TPP is required for carbohydrate
metabolism, and the brain depends on glucose metabolism for energy.
A proton readily dissociates from the C
that is between N and S in the thiazole ring
of TPP. The resulting carbanion (ylid) can attack the electron-deficient
keto carbon of pyruvate.
Lipoamide includes a dithiol that
undergoes oxidation and reduction.
The carboxyl group at the end of lipoic acid's
hydrocarbon chain forms an amide bond to the side-chain amino group of a
lysine residue of E2.
A long flexible arm, including hydrocarbon
chains of lipoate and the lysine R-group, links the dithiol of each lipoamide
to one of two lipoate-binding domains of each E2.
Lipoate-binding domains are themselves part of a flexible strand of E2
that extends out from the core of the complex.
The long flexible attachment allows lipoamide
functional groups to swing back and forth between E2 active sites
in the core of the complex and active sites of E1 & E3 in the
outer shell of the complex.
The E3 binding protein (that binds E3 to
E2) also has attached lipoamide that can exchange reducing equivalents
with lipoamide on E2.
FAD (Flavin Adenine Dinucleotide) is a derivative of the
B-vitamin riboflavin (dimethylisoalloxazine-ribitol). The flavin ring system
undergoes oxidation/reduction as shown below. Whereas NAD+ is
a coenzyme that reversibly binds to enzymes, FAD is a prosthetic group, that is permanently part of the
complex.
FAD
accepts and donates 2 electrons with 2 protons (2 H):
FAD
+ 2 e- + 2 H+ �� FADH2
Organic arsenicals are potent inhibitors of lipoamide-containing enzymes such as Pyruvate Dehydrogenase. These highly toxic compounds react with "vicinal" dithiols such as the functional group of lipoamide as shown below.
In the overall reaction, the acetic acid
generated is transferred to coenzyme A.
The final electron acceptor is NAD+.
The reaction proceeds as follows: |
|
The keto carbon of pyruvate reacts with the
carbanion of TPP on E1 to yield an addition compound. The electron-pulling
positively charged nitrogen of the thiazole ring promotes loss of CO2. What
remains is hydroxyethyl-TPP.
The hydroxyethyl carbanion on TPP of E1 reacts
with the disulfide of lipoamide on E2. What was the keto carbon of pyruvate is
oxidized to a carboxylic acid, as the disulfide of lipoamide is reduced to a
dithiol. The acetate formed by oxidation of the hydroxyethyl moiety is linked
to one of the thiols of the reduced lipoamide as a thioester (~).
The acetate is transferred from the thiol of
lipoamide to the thiol of coenzyme A, yielding acetyl CoA.
The reduced lipoamide swings over to the E3 active
site. Dihydrolipoamide is reoxidized to the disulfide, as 2 e- + 2 H+ are
transferred to a disulfide on E3 (disulfide interchange).
The dithiol on E3 is reoxidized as 2 e- + 2 H+
are transferred to FAD. The resulting FADH2 is reoxidized by electron transfer
to NAD+, to yield NADH + H+.
Acetyl CoA,
a product of the Pyruvate Dehydrogenase reaction, is a central compound in
metabolism. The "high energy" thioester linkage makes it an excellent
donor of the acetate moiety.
For example, acetyl CoA
functions as:
·
input to the Krebs Cycle, where the acetate moiety is
further degraded to CO2.
·
donor of acetate for synthesis of fatty acids, ketone bodies, and cholesterol.
Regulation of Pyruvate
Dehydrogenase complex.
Allosteric Regulation
Pyruvate dehydrogenase is a major regulatory point for entry of
materials into the citric acid cycle.. The enzyme is regulated allosterically
and by covalent modification.
E2 - inhibited by acetyl-CoA, activated by CoA-SH
E3 - inhibited by NADH, activated by NAD+.
ATP is an allosteric inhibitor of the complex, and AMP is an activator.
The activity of this key reaction is coordinated with the energy charge, the
[NAD+]/[NADH] ratio, and the ratio of acetylated to free coenzyme A.
Covalent Regulation
Part of the pyruvate dehydrogenase complex, pyruvate
dehydrogenase kinase, phosphorylates three specific E1 serine residues,
resulting in loss of activity of pyruvate dehydrogenase. NADH and acetyl-CoA
both activate the kinase. The serines are dephosphorylated by a specific enzyme
called pyruvate dehydrogenase phosphatase that hydrolyzes the phosphates from
the E1 subunit of the pyruvate dehydgrogenase complex. This has the
effect of activating the complex. The phosphatase is activated by Ca2+
and Mg2+. Because ATP and ADP differ in their affinities for Mg2+,
the concentration of free Mg2+ reflects the ATP/ADP ratio within the
mitochondrion. Thus, pyruvate dehydrogenase responds to ATP levels by being
turned off when ATP is abundant and further energy production is unneeded.
In mammalian tissues at rest, much less than half of the total pyruvate
dehydrogenase is in the active, nonphosphorylated form. The complex can be
turned on when low ATP levels signal a need to generate more ATP. The kinase
protein is an integral part of the pyruvate dehydrogenase complex, whereas the
phosphatase is but loosely bound.
Within the Krebs cycle, energy in the
form of ATP is usually derived from the breakdown of glucose, although fats and proteins can also be utilized as energy sources.
Since glucose can pass through cell membranes, it transports energy from one
part of the body to another. The Krebs cycle affects all types of life and is, as such, the
metabolic pathway within the cells. This pathway chemically converts
carbohydrates, fats, and proteins into carbon dioxide, and converts water into
serviceable energy.
The Krebs cycle is the second
stage of aerobic respiration, the first being glycolysis and last being the electron transport chain; the cycle is a series of
stages that every living cell must undergo in order to produce energy. The enzymes that cause each step of the process to occur are all located in the
cell's "power plant"; in animals, this power plant is the mitochondria;
in plants, it is the chloroplasts; and in microorganisms, it can be found in
the cell membrane. The Krebs cycle is also known as the citric acid cycle, because citric acid is the very first product
generated by this sequence of chemical conversions, and it is also regenerated
at the end of the cycle.
The pyruvate molecules produced during glycolysis
contains a lot of energy in the bonds between their molecules. In order
to use that energy, the cell must convert it into the form of ATP. To do so,
pyruvate molecules are processed through the Kreb Cycle, also known as the
citric acid cycle.
http://www.youtube.com/watch?v=7gR4s8ool1Y
(Kerbs Cycle as a drawing)
1. Prior to entering
the Krebs Cycle, pyruvate must be converted into acetyl CoA. This is achieved
by removing a CO2 molecule from pyruvate and then removing an electron to
reduce an NAD+ into NADH. An enzyme called coenzyme A is combined with the
remaini ow:
2. Citrate is formed
when the acetyl group from acetyl CoA combines with oxaloacetate from the
previous Krebs cycle.
3. Citrate is
converted into its isomer isocitrate.
4. Isocitrate is
oxidized to form the 5-carbon α-ketoglutarate. This step releases one
molecule of CO2 and reduces NAD+ to NADH2+.
5. The
α-ketoglutarate is oxidized to succinyl CoA, yielding CO2 and NADH2+.
The a-Ketoglutarate Dehydrogenase Complex is
Similar to pyruvate
dehydrogenase complex
Same coenzymes,
identical mechanisms
E1 - a-ketoglutarate
dehydrogenase (with TPP)
E2 – dihydrolipoyl succinyltransferase (with
flexible lipoamide prosthetic group)
E3 - dihydrolipoyl
dehydrogenase (with FAD)
6. Succinyl CoA
releases coenzyme A and phosphorylates ADP into ATP.
In the succinyl CoA
synthetase reaction, the thioester bond between HS-CoA and the succinyl group
is hydrolyzed.
Since it is a
rich in energy bond, the energy released is enough for synthesizing GTP from
GDP + (P).
This GTP is
equivalent, from the energetic point of view, to ATP. In fact, GTP can transfer
the (P) group to ADP to form ATP:
GTP + ADP ————–à GDP + ATP
Since ATP can
be produced from this reaction, without participation of the respiratory chain,
this process is called Substrate Level Phosphorylation (SLP) in contrast to the
Oxidative Phosphorylation (ATP synthesis using the energy released in the
Electron Transport Chain).
A few other
reactions in metabolism are also coupled with ATP synthesis without
participation of the respiratory chain. They are considered also SLP reactions.
7. Succinate is
oxidized to fumarate, converting FAD to FADH2.
The Succinate
Dehydrogenase Complex of several polypeptides, an FAD prosthetic group and
iron-sulfur clusters, embedded in the inner mitochondrial membrane. Electrons
are transferred from succinate to FAD and then to ubiquinone (Q) in electron
transport chain. Dehydrogenation is stereospecific; only the trans isomer is
formed
8. Fumarate is
hydrolized to form malate.
9. Malate is
oxidized to oxaloacetate, reducing NAD+ to NADH2+.
We are now back at the beginning of the Krebs Cycle.
Because glycolysis produces two pyruvate molecules from one glucose, each
glucose is processes through the kreb cycle twice. For each molecule of
glucose, six NADH2+, two FADH2, and two ATP.
The sum of all reactions in the citric
acid cycle is:
Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi
+ 2 H2O → CoA-SH + 3 NADH + 3 H+ + FADH2
+ GTP + 2 CO2
(the above reaction is equilibrated if Pi
represents the H2PO4- ion, GDP the GDP2-
ion and GTP the GTP3- ion).
Two carbons are oxidized to CO2, and the energy
from these reactions is stored in GTP,
NADH and FADH2. NADH and FADH2 are coenzymes (molecules that enable or enhance
enzymes) that store energy and are utilized in oxidative
phosphorylation.
Step |
Substrate |
Enzyme |
Reaction type |
Products/ |
Comment |
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1 |
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Acetyl CoA + |
CoA-SH |
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2 |
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3 |
Cis-Aconitate |
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·
Electrons
are also transferred to the electron
acceptor FAD,
forming FADH2.
·
At the end of
all cycles, the products are two GTP,
six NADH,
two FADH2, four CO2.
The
detailed chemical structures have very limited medical significance, but you
will find it very much easier to make sense of the other material in this
course if you take the trouble to learn them! It may be helpful to follow one
particular atom in acetyl CoA all the way round the cycle until it is lost as
carbon dioxide, and the coloured boxes are intended to assist this process.
http://www.youtube.com/watch?v=hw5nWB0xN0Y&feature=related
The oxidation of acetyl-CoA to CO2by
the TCA cycle is the central process in energy metabolism. However, the TCA cycle
also functions in biosynthetic pathways in which intermediates leave the cycle
to be converted primarily to glucose, fatty acids, or non-essential amino
acids. If TCA cycle anions are removed from the cycle they must be replaced to
permit its continued function. This process is termed anaplerosis.
Pyruvate carboxylase, which generates oxalacetate directly in the mitochondria,
is the major anaplerotic enzyme. Conversely, 4- and 5-carbon intermediates
enter the TCA cycle during the catabolism of amino acids. Because the TCA cycle
cannot fully oxidize 4- and 5-carbon compounds, these intermediates must be
removed from the cycle by a process termed cataplerosis.
Cataplerosis may be linked to
biosynthetic processes such as gluconeogenesis in the liver and kidney cortex,
fatty acid synthesis in the liver, and glyceroneogenesis in adipose tissue.
Cataplerotic enzymes present in many mammalian tissues include P-enolpyruvate
carboxykinase (PEPCK), glutamate dehydrogenase, aspartate aminotransferase, and
citrate lyase. In this review we have evaluated the roles of anaplerosis and
cataplerosis in whole body metabolism.
The TCA cycle is delicately balanced
between the inflow and output of intermediates for various metabolic processes.
The widely held view of the TCA cycle as a “metabolic furnace” needs
modification in light of information supporting its role in biosynthesis. The
cycle acts more as a traffic circle on a busy highway in which the flow of cars
into the circle must be balanced by the flow out or the entire traffic pattern
will be interrupted with disastrous consequences. In this essay we have
reviewed several metabolic situations in which the two key processes,
anaplerosis and cataplerosis, work together to ensure the appropriate balance of
carbon flow into and out of the TCA cycle. The beauty of this fundamental
biological mechanism is undeniable in its simplicity and ponderous in its
complexity.
INVESTIGATION OF BIOLOGICAL
OXIDATION, OXIDATIVE PHOSPHORYLATION AND
ATP SYNTHESIS. INHIBITORS AND
UNCOUPLERS OF OXIDATIVE PHOSPHORYLATION.
Bioenergetic
Bioenergetics is the part of biochemistry
concerned with the energy involved in making and breaking of chemical bonds in
the molecules
found in biological organisms.
Growth, development and metabolism
are some of the central phenomena in the study of biological organisms. The
role of energy is fundamental to such biological processes. The ability to harness
energy from a variety of metabolic pathways is a property of all living
organisms. Life
is dependent on energy transformations; living organisms
survive because of exchange of energy within and without.
In a living organism, chemical
bonds are broken and made as part of the exchange and transformation
of energy. Energy is available for work (such as mechanical work) or for other
processes (such as chemical synthesis and anabolic
processes in growth), when weak bonds are broken and stronger bonds are made.
The production of stronger bonds allows release of usable energy.
Living organisms obtain energy from
organic and inorganic materials. For example, lithotrophs
can oxidize minerals such as nitrates or forms of sulfur, such as
elemental sulfur, sulfites,
and hydrogen sulfide to produce ATP. In photosynthesis,
autotrophs
can produce ATP using light energy. Heterotrophs
must consume organic compounds. These are mostly carbohydrates,
fats, and proteins.
The amount of energy actually obtained by the organism is lower than the amount
present in the food; there are losses in digestion, metabolism, and thermogenesis.
Exergonic
and endergonic processes. Mechanism of energy releasing and storage in the
organism.
Exergonic refers to chemical reactions
that proceed spontaneously from reactants to products with the release of
energy. Endergonic reactions require energy input to proceed. Although the
terms are often used rather loosely, they are precisely defined thermodynamic
concepts based on changes in an entity called Gibbs free energy (G)
accompanying reactions. Reactions in which -G decreases are exergonic, and
those in which -G increases are endergonic. Exergonic reactions often involve
the breakdown of organic compounds found in food, whereas endergonic reactions
frequently entail synthesis of complicated molecules.
Biological metabolism contains many
examples of both types, and living organisms have developed elaborate
techniques for coupling the two.
Exergonic
reactions release free energy while endergonic reactions consume free energy
Although a negative -G indicates that energy must be
added to the system before a reaction will occur, it tells us nothing about the
rate at which it will progress. As is often the case, it may go very slowly if
substantial activation energy is required to start the reaction. Living
organisms have found a way around this problem by forming protein catalysts,
called enzymes, that effectively reduce the amount of activation energy needed,
and allow the reaction to proceed at a satisfactory rate. Enzymes do not affect
the free energy of the reaction, and will not enable reactions to proceed that
are not energetically feasible.
By coupling exergonic and endergonic
reactions, organisms are able to use the available energy in food they consume
to construct complex proteins, lipids, nucleic acids and carbohydrates needed
for their growth and development. A well-known example involves coupling the
formation of energy-rich adenosine triphosphate (ATP) from adenosine
diphosphate (ADP) and phosphate (an endergonic reaction), with the transfer of
hydrogen, removed from organic food materials, to oxygen (an exergonic
reaction). The process is called oxidative phosphorylation. Energy stored in
ATP may be used subsequently when the exergonic conversion of ATP back to ADP
and phosphate is coupled with the endergonic synthesis of a needed cellular
component.
What is macroergic bond? Examples of high energy compounds.
A bond in
chemical compounds, which acts as an energy accumulator. Macroergic bond
is present in some
phosphorus-containing compounds in living organisms, e. g.* adenosinetriphosphate.
Macroergic bonds
are formed as a result of complex biochemical processes
and break
up when energy is released. The processes are reversible and can be repeated.
High-energy phosphate can mean one of
two things:
·
The
phosphate-phosphate bonds formed when compounds such as adenosine diphosphate
and adenosine triphosphate are created.
·
The compounds
that contain these bonds, which include the nucleoside
diphosphates and nucleoside triphosphates, and the high-energy storage
compounds of the muscle, the phosphagens. When people speak of a high-energy phosphate
pool, they speak of the total concentration of these compounds with these
high-energy bonds.
·
High-energy phosphate bonds are pyrophosphate
bonds, acid anhydride
linkages formed by taking phosphoric acid derivatives and dehydrating
them. As a consequence, the hydrolysis of these bonds is exergonic under physiological conditions,
releasing energy.
Energy
released by high energy phosphate reactions |
|
Reaction |
ΔG
[kJ/mol] |
ATP + H2O
→ ADP + Pi |
-30.5 |
ADP + H2O
→ AMP + Pi |
-30.5 |
ATP + H2O
→ AMP + PPi |
-40.6 |
PPi
+ H2O → 2 Pi |
-31.8 |
AMP + H2O
→ A + Pi |
-12.6 |
Except for PPi → 2 Pi, these reactions
are, in general, not allowed to go uncontrolled in the human cell but are
instead coupled to other processes needing energy to drive them to completion.
Thus, high-energy phosphate reactions can:
·
provide energy to cellular processes,
allowing them to run;
·
couple processes to a particular
nucleoside, allowing for regulatory control of the process;
·
drive the reaction to the right,
by taking a reversible process and making it irreversible.
The one exception is of value because it allows a single
hydrolysis, ATP + 2H2O → AMP + PPi, to effectively
supply the energy of hydrolysis of two high-energy bonds, with the hydrolysis
of PPi being allowed to go to completion in a separate reaction. The
AMP is regenerated to ATP in two steps, with the equilibrium reaction ATP + AMP
↔ 2ADP, followed by regeneration of ATP by the usual means, oxidative phosphorylation or other
energy-producing pathways such as glycolysis.
Often, high-energy phosphate bonds are
denoted by the character '~'. In this "squiggle" notation, ATP
becomes A-P~P~P. The squiggle notation was invented by Fritz Albert Lipmann, who first
proposed ATP as the main energy transfer molecule of the cell, in 1941. It
emphasizes the special nature of these bonds.
Stryer states:
ATP is often called a high energy
compound and its phosphoanhydride bonds are referred to as high-energy bonds.
There is nothing special about the bonds themselves. They are high-energy
bonds in the sense that free energy is released when they are hydrolyzed,
for the reasons given above.
The term 'high energy' with respect to
these bonds can be misleading because the negative free energy change is not
due directly to the breaking of the bonds themselves. The breaking of these
bonds, as with the breaking of most bonds, is an endergonic step (i.e., it
absorbs energy, not releases it). The negative free energy change comes instead
from the fact that the bonds formed after hydrolysis-or the phosphorylation of
a residue by ATP-are lower in energy than the bonds present before hydrolysis
(this includes all of the bonds involved in the reaction, not just the
phosphate bonds themselves). This effect is due to a number of factors
including increased resonance stabilization and solvation
of the products relative to the reactants.
Besides the adenosine nucleotide phosphates,
uracil, cytosine and guanine phosphates occur, too:
UMP, UDP, UTP, CMP, CDP, CTP, GMP, GDP, GTP.
The triphosphate nucleosides of these compounds and those of ATP are
components of RNA. They are integrated into the polymer by splitting off
pyrophosphate ( = PP). The corresponding desoxyribose derivatives (dATP, dGTP,
dCTP....) are necessary for DNA synthesis, where dTTP is used instead of dUTP.
The terminal phosphate residues of all nucleoside di- and triphosphates are
equally rich in energy. The energy set free by their hydrolysis is used for
biosyntheses. They share the work equally: UTP is needed for the synthesis of
polysaccharides, CTP for that of lipids and GTP for the synthesis of proteins
and other molecules. These specificities are the results of the different
selectivities of the enzymes, that control each of these metabolic pathways.
ATP
formation
The Function of ATP
The ATP is used for many cell
functions including transport work moving substances across cell
membranes. It is also used for mechanical work, supplying the energy
needed for muscle contraction. It supplies energy not only to heart muscle (for
blood circulation) and skeletal muscle (such as for gross body movement), but
also to the chromosomes and flagella to enable them to carry out their many
functions. A major role of ATP is in chemical work, supplying the needed
energy to synthesize the multi-thousands of types of macromolecules that the
cell needs to exist.
ATP is also used as an on-off switch both to control
chemical reactions and to send messages. The shape of the protein chains that
produce the building blocks and other structures used in life is mostly
determined by weak chemical bonds that are easily broken and remade. These
chains can shorten, lengthen, and change shape in response to the input or withdrawal
of energy. The changes in the chains alter the shape of the protein and can
also alter its function or cause it to become either active or inactive.
The ATP molecule can bond to
one part of a protein molecule, causing another part of the same molecule to
slide or move slightly which causes it to change its conformation, inactivating
the molecule. Subsequent removal of ATP causes the protein to return to its
original shape, and thus it is again functional. The cycle can be repeated
until the molecule is recycled, effectively serving as an on and off switch.
Both adding a phosphorus (phosphorylation) and removing a phosphorus from a
protein (dephosphorylation) can serve as either an on or an off switch.
How is ATP Produced?
ATP is manufactured as a result of
several cell processes including fermentation, respiration and photosynthesis.
Most commonly the cells use ADP as a precursor molecule and then add a
phosphorus to it. In eukaryotes this can occur either in the soluble portion of
the cytoplasm (cytosol) or in special energy-producing structures called
mitochondria. Charging ADP to form ATP in the mitochondria is called chemiosmotic
phosphorylation. This process occurs in specially constructed chambers
located in the mitochondrion’s inner membranes.
ATP-synthase
converts ADP into ATP, a process called charging.
Adenosine triphosphate (ATP)
is an organic molecule which stores energy used to carry out life processes. ATP
is made of an adenine nucleoside, ribose sugar, and three phosphate groups. The
high energy bonds between phosphate groups are broken when hydrolyzed, thus
releasing energy in the system. Either one or two phosphate groups can break
off, releasing Gibb's free energy,
which can then be used to drive other reactions.
The molecular structure of ATP which is formed from
a adenine nucleoside, ribose sugar, and three phosphate groups
ATP can be formed from bonding either
adenosine monophosphate (AMP) and two inorganic phosphate groups (PPi)
together or by bonding adenosine diphosphate (ADP) and one inorganic phosphate
group (Pi) together. Energy is required to bond the adenosine to the
phosphate groups, making it an endergonic reaction. The energy used to bond the
two molecules together is then stored within covalent bonds between phosphate
groups in ATP. ATP
can be formed through two different endergonic processes, either through substrate-level
phosphorylation or chemiosmosis.
ATP is needed
- as a source
of energy for biochemical syntheses
- for transport processes
(active transport) and
- for mechanical work
like movements (ciliar movements, plasma currents etc.)
How the Hydrolysis of ATP Performs Work
•The bonds between the phosphate groups of ATP’s
tail can be broken by hydrolysis
•Energy is released from ATP when the terminal
phosphate bond is broken
•This release of energy comes from the chemical
change to a state of lower free energy, not from the phosphate bonds themselves
•The three types of cellular work (mechanical,
transport, and chemical) are powered by the hydrolysis of ATP
•In the cell, the energy from the exergonic
reaction of ATP hydrolysis can be used to drive an endergonic reaction
•Overall, the coupled reactions are exergonic
ATP + H2O →
ADP + Pi
Releases -30.5 kJ/mol= ΔG˚ (when
one phosphate group breaks off)
ATP + H2O →
AMP + PPi
Releases -45.6 kJ/mol= ΔG˚ (when two
phosphate groups break off)
The Regeneration
of ATP
•ATP is a renewable resource that is regenerated by addition of a
phosphate group to adenosine diphosphate (ADP)
•The energy to phosphorylate ADP comes from catabolic reactions in the
cell
•The ATP cycle is a revolving door through which energy passes during
its transfer from catabolic to anabolic pathways
http://www.youtube.com/watch?v=_PgjsfY71AM&feature=related
http://www.youtube.com/watch?v=YndC0gS3t6M&feature=related
Substrate-level and oxidative phosphorylation
Substrate-level phosphorylation is
a type of metabolic reaction that results in the formation of adenosine
triphosphate (ATP) or guanosine
triphosphate (GTP) by the direct transfer and
donation of a phosphoryl (PO3)
group to adenosine
diphosphate (ADP) or guanosine
diphosphate (GDP) from a phosphorylated reactive
intermediate. Note that the phosphate group does
not have to come directly from the substrate. By convention, the phosphoryl
group that is transferred is referred to as a phosphate group.
An alternative way to create ATP is
through oxidative
phosphorylation, which takes place during the
process of cellular
respiration, in addition to the substrate-level
phosphorylation that occurs during glycolysis and the Krebs cycle. During
oxidative phosphorylation, NADH is oxidized to NAD+, yielding 2.5
ATPs, and FADH2 yields 1.5 ATPs when it is oxidized. Oxidative
phosphorylation uses an electrochemical or chemiosmotic gradient of protons (H+)
across the inner mitochondrial membrane to generate ATP from ADP, which is a
key difference from substrate-level phosphorylation.
Unlike oxidative phosphorylation, oxidation
and phosphorylation
are not coupled in the process of substrate-level phosphorylation, although
both types of phosphorylation result in the formation of ATP and reactive
intermediates are most often gained in course of oxidation processes in catabolism.
However, usually most of the ATP is generated by oxidative phosphorylation in
aerobic or anaerobic respiration. Substrate-level phosphorylation serves as
fast source of ATP independent of external electron acceptors and respiration.
This is the case for example in human erythrocytes, which have no mitochondria, and in
the muscle during oxygen depression.
The main part of substrate-level
phosphorylation occurs in the cytoplasm of cells as part of glycolysis
and in mitochondria as part of the Krebs Cycle under both aerobic
and anaerobic
conditions. In the pay-off phase of
glycolysis, two ATP are produced by
substrate-level phosphorylation: two and only two 1,3-bisphosphoglycerate
are converted to 3-phosphoglycerate by transferring a
phosphate group to ADP by a kinase; two phosphoenolpyruvate are converted to pyruvate
by the transfer of their phosphate groups to ADP by another kinase. The first
reaction occurs after the generation of 1,3-bisphosphoglycerate from
3-phosphoglyceraldehyde and an organic phosphate via a dehydrogenase.
ATP is generated in a following separate step (key
difference from oxidative phosphorylation) by transfer of the high-energy
phosphate on 1,3-bisphosphoglycerate to ADP via the enzyme phosphoglycerate kinase,
generating 3-phosphoglycerate. As ATP is formed of a former inorganic phosphate
group, this step leads to the energy yield of glycolysis. The second
substrate-level phosphorylation occurs later by means of the reaction of
phosphenolpyruvate (PEP) to pyruvate via the pyruvate kinase. This reaction regenerates the ATP
that has been used in the preparatory phase of glycolysis to activate glucose
to glucose-6-phosphate and fructose-6-phosphate to fructose-1,6-bisphosphate,
respectively.
ATP can be generated by
substrate-level phosphorylation in the mitochondrial matrix, a pathway that is
independent from the protonmotive force, pmf. In the mitochondrial matrix there
are two reactions capable of substrate-level phosphorylation: the mitochondrial
phosphoenolpyruvate carboxykinase (PEPCK), and the succinate-CoA ligase (SUCL
or succinate thiokinase or succinyl-CoA synthetase). Mitochondrial PEPCK is
thought to participate in the transfer of the phosphorylation potential from
the matrix to cytosol and vice versa. The enzyme is a heterodimer, being composed
of an invariant alpha subunit encoded by SUCLG1, and a substrate-specific beta
subunit, encoded by either SUCLA2 or SUCLG2. This dimer combination results in
either an ADP-forming succinate-CoA ligase (A-SUCL, EC 6.2.1.5) or a
GDP-forming succinate-CoA ligase (G-SUCL, EC 6.2.1.4). The ADP-forming
succinate-CoA ligase is potentially the only matrix enzyme generating ATP in
the absence of a pmf, capable of maintaining matrix ATP levels under
energy-limited conditions, such as transient hypoxia.
Another form of substrate-level phosphorylation is
also seen 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.
Apart from this substrate-level phosphorylation can
also be observed in fermentation,
for example, heterolactic
fermentation, butyric acid fermentation, and
propanoic acid fermentation.
The modern
views on the biological oxidation
Biological oxidation is that oxidation which occurs in
biological systems to produce energy.
Oxidation can occur by:
1-Addition of oxygen (less common)
2-Removal of hydrogen (common)
3-Removal of electrons (most common)
Electrons are not stable in the free state, so their
removal form a substance (oxidation) must be accompanied by their
acceptance by another substance (reduction) hence the reaction is called
oxidation-reduction reaction or redox reaction and the involved enzymes are
called oxido-reductases
Redoxpotential
It
is the affinity of a substance to accept electrons i.e. it is the potential for
a substance to become reduced. Hydrogen
has the lowest redoxpotential (-0.42 volt), while oxygen has the highest
redoxpotential (+0.82 volt). The redoxpotentials of all other substances lie
between that of hydrogen and oxygen.
Electrons are transferred from substances with low
redoxpotential to substances with higher redoxpotential.This transfer of
electrons is an energy yielding process and the amount of energy liberated
depends on the redoxpotential difference between the electron donor and acceptor.
Oxido-reductases
These enzymes catalyze oxidation-reduction reactions.
They are classified
into five groups:
1-oxidases. 2-aerobic
dehydrogenises.
3-anaerobic
dehydrogenises.
4-hydroperoxidasesand
5-oxygenases.
1. Oxidases
An oxidase is any enzyme that catalyzes
an oxidation-reduction
reaction involving molecular oxygen (O2) as the electron acceptor. In these
reactions, oxygen is reduced to water (H2O) or hydrogen
peroxide (H2O2). The oxidases are a subclass
of the oxidoreductases.
OXIDATION-REDUCTION
OR REDOX REACTIONS
2. Aerobic
Dehydrogenases(FlavoproteinLinked Oxidases).
The
coenzyme of aerobic dehydrogenasesmay be:
•FMN (Flavinadenine
mononucleotide) as in L-amino acid oxidase.
•FAD (Flavinadenine
dinucleotide) as in D-amino acid oxidase, xanthineoxidase,
aldehydedehydrogenaseand glucose oxidase.
3. Anaerobic Dehydrogenases
Anaerobic
dehydrogenasesare further classified according to their coenzymes into:
•NAD+linked anaerobic
dehydrogenasese.g.
a)Cytoplasmicglycerol-3-phosphate
dehydrogenase
b)Isocitratedehydrogenase.
c)Malatedehydrogenase.
d)β-HydroxyacylCoAdehydrogenase.
e)β-Hydroxybutyrate
dehydrogenase.
•NADP
linked anaerobic dehydrogenasese.g.
a)Glucose-6-phosphate
dehydrogenase.
b)Malicenzyme.c)Cytoplasmicisocitratedehydrogenase
•FAD
linked anaerobic dehydrogenasese.g.
a)Succinatedehydrogenase.
b)Mitochondrial
glycerol-3-phosphate dehydrogenase.
c)Acy1
CoAdehydrogenase.
4. Hydroperoxidases
These enzymes use hydrogen peroxide (H2O2) as substrate changing it into
water to get rid of its harmful effects.
They are further classified into peroxidasesand catalases.
•Peroxidases:
These enzymes need a reduced substrate as hydrogen donorperoxidase
H2O2
+ XH2 (reduced substrate) ----------→ X(oxidized substrate)+ 2H2O Example:
-Glutathione
peroxidasegets rid of H2O2from red cells to protect them from haemolysis
Glutathione
Peroxidase
H2O2
+ 2 G-S H -----------→2H2O + G-S-S-G
•Catalases:These
enzymes act on 2 molecules of hydrogen peroxide; one molecule is hydrogen donor
& the other molecule is hydrogenaccepetor.
2H2O2
+ catalase---------→ 2H2O + O2
Hydrogen
peroxide is continuously produced by the action of aerobic dehydrogenasesand
some oxidases. It is also produced by action of superoxide dismutase on
superoxide (O•2). It is removed by the action of peroxidasesand catalasesto protect
cells against its harmful effects.
5. Oxygenases
These enzymes catalyze direct incorporation (addition) of oxygen into
substrate.
They are further classified into dioxygenasesand monooxygenases.
A. Dioxygenases(true
oxgenases)
These
enzymes catalyze direct incorporation of two atoms of oxygen molecule into
substrate e.g. tryptophan pyrrolase, homnogentisicacid dioxygenase,
carotenaseand β-hydroxyanthranilicacid dioxygenase.
Dioxygenase
A + O2 → AO2
B.
Mono-oxygenases(pseudo-oxygenases; hydroxylases; mixed: function oxygenases)
AH + O2+ XH2 ------→A-OH + H2O + X
Fuctionsof
cytochromeP450
Functions
of microsomalcytochromeP450
1-It
is important for detoxicationof xenobioticsby hydroxylation. e.g.
insecticides,carcinogens,mutagensand drugs.
2-It
is also important for metabolism of some drugs by hydroxylation e.g.
morphine, aminopyrine, benzpyrineand aniline.
drug-H
+ O2+ XH2 drug-OH + H2O + X→ drug-OH + H2O + X
Function
of mitochondrial cytochromeP450
1-It
has a role in biosynthesis of steroid hormones from cholesterol in adrenal
cortex, testis, ovary and placenta by hydroxylation
2-It
has a role in biosynthesis of bile acids from cholesterol in the liver by
hydroxylation at C26 by 26 hydroxylase.
3-It
is important for activation of vitamin D
CytochromeP450
It
is a group of hydroxylaseswhich are collectively referred to as cytochromeP450.
They
are so called because their reduced forms exhibit an intense absorption band at
wavelength 450 nm when complexedto carbon monoxide.
They
are conjugated protein containing haeme(haemoproteins).
According
to their intracellular localization they may be classified into:
•MicrosomalcytochromeP450.
It
is present mainly in the microsomes of
liver cells. It represents about 14% of the microsomalfraction of liver cells.
•Mitochondrial
cytochromeP450.
It
is present in mitochondria of many tissues but it is particularly abundant in
liver and steroidogenictissues as adrenal cortex, testis, ovary, placenta and
kidney.
Tissue Respiration.
Tissue respiration is the release of
energy, usually from glucose, in the tissues of all animals, green plants,
fungi and bacteria. All these living things require energy for other processes
such as growth, movement, sensitivity, and reproduction.
The most efficient form of
respiration is aerobic respiration: this requires oxygen. When oxygen is not
available, some organisms can respire anaerobically i.e. without air or oxygen.
Yeast can respire in both ways. Yeast gets more energy from aerobic respiration,
but when it runs out of oxygen it does not die. It can continue to respire
anaerobically, but it does not get so much energy from the sugar. Yeast
produces ethanol (alcohol) when it respires anaerobically and ultimately the
ethanol will kill the yeast.
We can respire in both ways too. Normally we use
oxygen, but when we are running in a race, we may not get enough oxygen into
our blood, so our muscles start to respire anaerobically. Unlike yeast we
produce lactic acid. Of course if we produced alcohol in our muscles it would
make us drunk! Fine thing if you are running away from a predator and you end
up drunk! Making lactic acid is not much better. Lactic acid causes cramp.
Glucose +
Oxygen = Carbon Dioxide + Water + Energy
This word equation means: “sugar and oxygen are turned
into carbon dioxide and water releasing energy”. You must memorise the word
equation (and the balanced chemical equation if you want a grade A, B or C).
Get help memorising the equations
Glucose =
Carbon Dioxide + Ethanol + Energy
This word equation means: “glucose is turned into
carbon dioxide and ethanol releasing energy”. You must memorise this word equation.
Composition
of respiratory chain
NADH dehydrogenase (EC 1.6.5.3) (also
referred to as "NADH:quinone reductase" or "Complex I") is
an enzyme located in the
inner mitochondrial membrane
that catalyzes the transfer of electrons from NADH to coenzyme Q (CoQ). It is
the "entry enzyme" of oxidative
phosphorylation in the mitochondria.
NADH Dehydrogenase is the first
enzyme (Complex I) of the mitochondrial electron
transport chain. There are three energy-transducing
enzymes in the electron transport chain - NADH dehydrogenase (Complex I), Coenzyme Q –
cytochrome c reductase (Complex III), and cytochrome c
oxidase (Complex IV). NADH dehydrogenase is the largest
and most complicated enzyme of the electron transport chain..
The reaction
of NADH dehydrogenase is:
In this process, the complex translocates four protons across the
inner membrane per molecule of oxidized NADH, helping to
build the electrochemical
potential used to produce ATP.
The reaction can be reversed - referred to as aerobic
succinate-supported NAD+ reduction - in the presence of a high
membrane potential, but the exact catalytic mechanism remains unknown.
Complex I may have a role in triggering apoptosis. [5] In fact,
there has been shown to be a correlation between mitochondrial activities and programmed
cell death (PCD) during somatic embryo
development.
All redox reactions take place in the extramembranous
portion of NADH dehydrogenase. NADH initially binds to NADH dehydrogenase, and
transfers two electrons to the flavin
mononucleotide (FMN) prosthetic group of complex I,
creating FMNH2. The electron acceptor - the isoalloxazine ring - of
FMN is identical to that of FAD. The electrons are then
transferred through the second prosthetic group of NADH dehydrogenase via a
series of iron-sulfur (Fe-S) clusters, and finally to coenzyme Q
(ubiquinone). This electron flow causes four hydrogen ions to be pumped out of
the mitochondrial matrix. Ubiquinone (CoQ)
accepts two electrons to be reduced to ubiquionol (CoQH2).
FADH dehydrogenase
In biochemistry, flavin
adenine dinucleotide (FAD) is a redox cofactor
involved in several important reactions in metabolism.
FAD can exist in two different redox states, which it converts between by
accepting or donating electrons. The molecule consists of a riboflavin moiety
(vitamin B2) bound to the phosphate group of an ADP
molecule. The flavin group is bound to ribitol, a sugar
alcohol, by a carbon-nitrogen bond, not a glycosidic bond.
Thus, riboflavin is not technically a nucleotide; the name flavin adenine
dinucleotide is a misnomer.[1]
FAD can be reduced to FADH2, whereby it accepts
two hydrogen atoms (a net gain of two electrons):
FAD (fully oxidized form, or quinone
form) accepts two electrons and two protons to become FADH2
(hydroquinone form). FADH2 can then be oxidized to the semireduced
form (semiquinone) FADH by donating one electron and one proton. The semiquinone
is then oxidized once more by losing an electron and a proton and is returned
to the initial quinone form (FAD).
FAD is an aromatic ring system, whereas FADH2 is not. This means that FADH2
is significantly higher in energy, without the stabilization that aromatic
structure provides. FADH2 is an energy-carrying molecule, because,
if it is oxidized, it will regain aromaticity and release all the energy
represented by this stabilization.
The primary biochemical role of FADH2 in eukaryotes is to carry
high-energy electrons used for oxidative
phosphorylation. Its hydrogens remain in the
mitochondrial matrix, whilst FAD is tightly bound to a dehydrogenase enzyme
i.e. the second protein complex in the oxidative phosphorylation chain. FAD is
a prosthetic group in the
enzyme complex succinate
dehydrogenase (complex II) that oxidizes succinate to fumarate in the
eighth step of the citric acid cycle. The high-energy
electrons from this oxidation are stored momentarily by reducing FAD to FADH2.
FADH2 then reverts to FAD, sending its two high-energy electrons
through the electron
transport chain; the energy in FADH2 is
enough to produce 1.5 equivalents of ATP[2] by oxidative
phosphorylation. Another metabolic source of FADH2 is beta oxidation, where FAD
serves as a coenzyme to acyl CoA
dehydrogenase. A flavoprotein
is a protein that contains a flavin moiety, this may be in the form of FAD or
FMN (Flavin
mononucleotide) . There are many flavoproteins
besides components of the succinate dehydrogenase complex, including α-ketoglutarate dehydrogenase
and a component of the pyruvate
dehydrogenase complex.
Ubiquinones
These molecules are also known as
coenzyme Q or mitoquinones. They are involved in electron transport in
mitochondrial preparations playing an essential role in the oxidation of
succinate or NADH via the cytochrome system. They serves not only as a coenzyme
but also, in their reduced forms, as antioxidants. They are synthesized de novo
in all animal tissues and cannot thus be regarded as vitamins. Ubiquinones are
present in all aerobic organisms, plants, animals (the name ubiquinone was
proposed with reference to their ubiquitous occurrence) and bacteria, but are
absent from Gram-positive eubacteria and the archaebacteria. They were discovered by the Morton's group
in animal fat but their quinonoid structure was revealed by Crane two years
later in extracts from beef heart mitochondria.
The compound had a 2,3-dimethoxy-5-methylbenzoquinone
nucleus and a side chain of 10 isoprenoid units and
was referred to as coenzyme Q 10 . Later, homologues with 6, 7, 8 and 9 units
were isolated from other organisms, bacteria or higher organisms. The main form
in man has 10 units but in rat has 9 units. Another system of nomenclature is
used: ubiquinone(x) in which x designates the total number of carbon atoms in
the side chain, it can be a multiple of 5.
Ubiquinones accept one electron and
are transformed into semiquinone radicals (UQH°) or two electrons to give
ubiquinol (UQH2)
Coenzyme Q is reducible by sodium dithionite or
borohydride to its hydroquinone form, and can in turn be reoxidized to the
quinone by Ag2O or more slowly by oxygen. The absorption spectra of the two
forms are shown below. The quinone form has a strong absorption band at 275 nm
which disappears in the reduced form.
Cytochromes
are, in general, membrane-bound (i.e. inner mitochondrial membrane) hemeproteins
containing heme
groups and are primarily responsible for the generation of ATP
via electron transport.
They are found either as monomeric proteins
(e.g., cytochrome c)
or as subunits
of bigger enzymatic complexes that catalyze redox
reactions.
Cytochromes were initially described in 1884 by
MacMunn as respiratory pigments (myohematin or histohematin).[1]
In the 1920s, Keilin
rediscovered these respiratory pigments and named them the cytochromes, or
“cellular pigments”, and classified these heme proteins, on the basis of the
position of their lowest energy absorption band in the reduced state, as
cytochromes a (605 nm), b (~565 nm), and c
(550 nm). The UV-visible spectroscopic signatures of hemes are still used
to identify heme type from the reduced bis-pyridine-ligated state, i.e., the
pyridine hemochrome method. Within each class, cytochrome a, b,
or c, early cytochromes are numbered consecutively, e.g. cyt c,
cyt c1, and cyt c2, with more recent
examples designated by their reduced state R-band maximum, e.g. cyt c559.[2]
The heme
group is a highly-conjugated ring system (which allows its electrons
to be very mobile) surrounding a metal ion, which readily interconverts between
the oxidation states. For many cytochromes, the metal ion present is that of iron,
which interconverts between Fe2+ (reduced) and Fe3+
(oxidised) states (electron-transfer
processes) or between Fe2+ (reduced) and Fe3+ (formal,
oxidized) states (oxidative processes). Cytochromes are, thus, capable of
performing oxidation and reduction.
Because the cytochromes (as well as other complexes) are held within membranes
in an organized way, the redox
reactions are carried out in the proper sequence for maximum efficiency.
In the process of oxidative phosphorylation,
which is the principal energy-generating process undertaken by organisms, other
membrane-bound and -soluble complexes and cofactors are involved
in the chain of redox reactions, with the additional net effect that protons (H+)
are transported across the mitochondrial inner membrane. The resulting transmembrane proton gradient
([protonmotive force]) is used to generate ATP, which is
the universal chemical energy currency of life. ATP is consumed to drive cellular
processes that require energy (such as synthesis of macromolecules, active
transport of molecules across the membrane, and assembly of flagella).
Several kinds of cytochrome exist and
can be distinguished by spectroscopy, exact
structure of the heme group, inhibitor sensitivity, and reduction potential.
Three types of cytochrome are
distinguished by their prosthetic groups:
Type |
Prosthetic group |
The definition of cytochrome c is not defined
in terms of the heme group. There is no "cytochrome e," but there is
a cytochrome f, which is
often considered a type of cytochrome c.
In mitochondria and chloroplasts, these
cytochromes are often combined in electron
transport and related metabolic pathways:
Cytochromes |
Combination |
a and a3 |
Cytochrome
c oxidase ("Complex IV") with
electrons delivered to complex by soluble cytochrome c
(hence the name) |
b and c1 |
Coenzyme Q - cytochrome c reductase
("Complex III") |
b6 and f |
A completely distinct family of cytochromes is known
as the cytochrome
P450 oxidases, so named for the characteristic Soret peak formed by
absorbance of light at wavelengths near 450 nm when the heme iron is
reduced (with sodium dithionite) and
complexed to carbon monoxide. These
enzymes are primarily involved in steroidogenesis
and detoxification.
Respiratory chain
The
system of mitochondrial enzymes and redox carrier molecules which ferry
reducing equivalents from substrates to oxygen are collectively known as the electron
transport system, or the respiratory chain. This system captures the free energy available
from substrate oxidation so that it may later be applied to the synthesis of
ATP. Many respiratory chain components were first identified in crude homogenates
through their spectral properties, which frequently change when a carrier is
oxidised or reduced. Fractionation of mitochondria in the presence of mild
detergents or chaotropic salts dissected the respiratory chain into four large
multi-subunit complexes containing the principal respiratory carriers, named Complex
1 to Complex 4.
These
substantial protein "icebergs" float in the sheet of inner membrane lipids, often
presenting one face to the mitochondrial matrix
and another to the inter - membrane space.
Many of their components have now been isolated in a relatively pure form.
Other membrane bound enzymes such as the energy linked transhydrogenase (ELTH)
are also present which fulfil ancillary roles.
The main components participate in the approximate
order of their redox potentials, and the
bulky complexes are linked by low molecular weight mobile carriers which ferry
the reducing equivalents from one complex to the next. Except for succinate
dehydrogenase (complex 2) all these complexes pump protons from the matrix
space into the cytosol as they transfer reducing equivalents (either hydrogen
atoms or electrons) from one carrier to the next. The diagram above shows the
flow of reducing equivalents in purple, and movement of the positively charged
protons in red. Proton pumping is an arduous task which creates substantial pH
and electrical gradients across the mitochondrial inner membrane. These protons
eventually re-enter the matrix space via the F1 ATPase, driving the
synthesis of ATP as they return.
The number of protons and the number of positive
charges crossing the inner membrane need not necessarily agree for each
individual transmembrane protein, although the accounts must balance for the
whole ensemble. This discrepancy is illustrated on the diagram above, and is
explained in greater detail below.
Electrons flow through the electron
transport chain to molecular oxygen; during this flow, protons are moved across
the inner membrane from the matrix to the intermembrane space. This model for
ATP synthesis is called the chemiosmotic mechanism, or Mitchell
hypothesis. Peter Mitchell, a British biochemist, essentially by himself and in
the face of contrary opinion, proposed that the mechanism for ATP synthesis
involved the coupling between chemical energy (ATP) and osmotic potential (a
higher concentration of protons in the intermembrane space than in the matrix).
The inner membrane of the mitochondrion is tightly packed with cytochromes and
proteins capable of undergoing redox changes. There are four major
protein-membrane complexes.
Complex I and Complex II direct
electrons to coenzyme Q. Complex I, also called NADH-coenzyme Q reductase,
accepts electrons from NADH. The NADH releases a proton and two electrons. The
electrons flow through a flavoprotein containing FMN and an iron-sulfur
protein. First, the flavin coenzyme (flavin mononucleotide) and then the
iron-sulfur center undergo cycles of reduction and then oxidation, transferring
their electrons to a quinone molecule, coenzyme Q
(see Figure ).
Complex I is capable of transferring protons from the
matrix to the intermembrane space while undergoing these redox cycles. One
possible source of the protons is the release of a proton from NADH as it is
oxidized to NAD, although this is not the only explanation. Apparently,
conformational changes in the proteins of Complex I also are involved in the
mechanism of proton translocation during electron transport.
|
Complex II, also known as succinate-coenzyme
Q reductase, accepts electrons from succinate formed during
the TCA cycle. Electrons flow from succinate to FAD (the flavin-adenine
dinucleotide) coenzyme, through an iron-sulfur protein and a cytochrome b550
protein (the number refers to the wavelength where the protein absorbs), and to
coenzyme Q. No protons are translocated by Complex II. Because translocated
protons are the source of the energy for ATP synthesis, this means that the
oxidation of a molecule of FADH2 inherently leads to less ATP
synthesized than does the oxidation of a molecule of NADH. This experimental
observation also fits with the difference in the standard reduction potentials
of the two molecules. The reduction potential of FAD is -0.22 V, as opposed to
-0.32 V for NAD.
Coenzyme Q is capable of accepting either one or
two electrons to form either a semiquinone or hydroquinone
form. Coenzyme Q is not bound to a protein; instead it is a mobile electron
carrier and can float within the inner membrane, where it can transfer
electrons from Complex I and Complex II to Complex III.
Complex III is also known as coenzyme
Q-cytochrome c reductase. It accepts electrons from reduced coenzyme
Q, moves them within the complex through two cytochromes b, an iron-sulfur
protein, and cytochrome c1. Electron flow through Complex II
transfers proton(s) through the membrane into the intermembrane space. Again,
this supplies energy for ATP synthesis. Complex III transfers its electrons to
the heme group of a small, mobile electron transport protein, cytochrome
c.
Cytochrome c transfers its electrons
to the final electron transport component, Complex IV, or cytochrome
oxidase. Cytochrome oxidase transfers electrons through a
copper-containing protein, cytochrome a, and cytochrome a3, and
finally to molecular oxygen. The overall pathway for electron
transport is therefore:
|
||
|
|
The number n is a fudge factor
to account for the fact that the exact stoichiometry of proton transfer isn't
really known. The important point is that more proton transfer occurs from NADH
oxidation than from FADH2 oxidation.
A theory postulated by the biochemist
Peter Mitchell in 1961 to describe ATP synthesis by way of a proton
electrochemical coupling is called chemiosmotic hypothesis.
Accordingly, hydrogen ions (protons) are pumped from the
mitochondrial matrix to the intermembrane space via the hydrogen carrier
proteins while the electrons are transferred along the electron transport chain
in the mitochondrial inner membrane. As the hydrogen ions accumulate in the
intermembrane space, an energy-rich proton gradient is established. As the
proton gradient becomes sufficiently intense the hydrogen ions tend to diffuse
back to the matrix (where hydrogen ions are less) via the ATP synthase (a
transport protein). As the hydrogen ions diffuse (through the ATP synthase)
energy is released which is then used to drive the conversion of ADP to ATP (by
phosphorylation).
Chemiosmotic Hypothesis in a simple form
In the 1960s, ATP
was known to be the energy currency of life, but the mechanism by which ATP was
created in the mitochondria was assumed
to be by substrate-level
phosphorylation. Mitchell's chemiosmotic hypothesis
was the basis for understanding the actual process of oxidative
phosphorylation. At the time, the biochemical
mechanism of ATP synthesis by oxidative phosphorylation was unknown.
Mitchell realised that the movement
of ions across an electrochemical
membrane potential could provide the energy needed to
produce ATP. His hypothesis was derived from information that was well known in
the 1960s. He knew that living cells had a membrane potential;
interior negative to the environment. The movement of charged ions across a
membrane is thus affected by the electrical forces (the attraction of positive
to negative charges). Their movement is also affected by thermodynamic
forces, the tendency of substances to diffuse from regions
of higher concentration. He went on to show that ATP synthesis was coupled to
this electrochemical
gradient.
His hypothesis was confirmed by the
discovery of ATP synthase, a
membrane-bound protein that uses the potential energy of the electrochemical
gradient to make ATP.
The passage back occurs via a specific proton
channel. This passage is coupled to ATP-synthesis, using the potential energy
of the proton gradient for the formation of the third phosphate bond of ATP.
We can now calculate the end result of glucose degradation:
the oxidation is coupled to a decrease of the free energy; 686 kcal/mol (= 2881
kJ/mol) are obtained by the complete oxidation of glucose. How much of this energy can the cell
use?
1.
Six mol ATP per mol glucose are
generated (substrate chain phosphorylation). This is because all steps after
the breaking down of fructose-1,6-phosphate have to be counted twice (once for
each of the two resulting C3 molecules), so it is 3 x 2 ATPs. Of
these six ATPs, two are needed to start glycolysis. That leaves four.
2.
During the course of glycolysis up to
acetyl-CoA, 2 x 2 NADH + H+ are generated. An additional 3 x 2 NADH
+ H+ and 1 x 2 FADH2 are produced in the citric acid
cycle. One NADH + H+ gives three, one FADH2 two ATPs when
fed into the respiratory chain. This sums up to 34 ATPs plus the 4 ATPs of
glycolysis. A total of 38 mol ATP are thus gained by the cell's degradation of
one mol glucose. Since each energy-rich bond of ATP contains 7.3 kcal/mol (=
-30.6 kJ/mol), the 38 ATP equal 277 kcal/mol (ca 1163 kJ/mol). This is 40.6% of
the theoretically possible gain. The other 59.4 percent are set free as heat.
This is a very high percentage compared to the gain of technical machines like
steam or petrol engines that is around or below 20 percent.
The ATP synthase enzymes have been remarkably
conserved through evolution. The bacterial enzymes are essentially the same in
structure and function as those from mitochondria of animals, plants and fungi,
and the chloroplasts of plants. The early ancestory of the enzyme is seen in
the fact that the Archaea have an enzyme which is clearly closely related, but
has significant differences from the Eubacterial branch. The H+-ATP-ase
found in vacuoles of the eukaryote cell cytoplasm is similar to the archaeal
enzyme, and is thought to reflect the origin from an archaeal ancestor.
In most systems, the ATP synthase sits in the membrane
(the "coupling" membrane), and catalyses the synthesis of ATP from
ADP and phosphate driven by a flux of protons across the membrane down the
proton gradient generated by electron transfer. The flux goes from the
protochemically positive (P) side (high proton electrochemical potential) to
the protochemically negative (N) side. The reaction catalyzed by ATP synthase
is fully reversible, so ATP hydrolysis generates a proton gradient by a
reversal of this flux. In some bacteria, the main function is to operate in the
ATP hydrolysis direction, using ATP generated by fermentative metabolism to
provide a proton gradient to drive substrate accumulation, and maintain ionic
balance.
ADP + Pi + nH+P <=> ATP + nH+N
·
the free energy change (DG) associated with synthesis of
ATP under cellular conditions (the free energy required)
Because the structures seen in EM, the subunit
composition, and the sequences of the subunits appeared to be so similar, it
had been assumed that the mechanisms, and hence the stoichiometries, would be the
same. In this context, the evidence suggesting that the stoichiometry of H+/ATP
(n above) varied depending on system was surprising. Values based on measure
ATP/2e- ratios, and H+/2e- ratios had
suggested that n was 3 for mitochondria, and 4 for chloroplasts, but these
values were based on the assumption of integer stoichiometries. Although all
the F1F0-type ATP-synthases likely had a common origin,
both the assumption that the stoichiometries are the same, and that n is
integer, are called into question by emerging structural data (see below).
The
structure of the soluble (F1) portion of the ATP synthase from beef
heart mitochondria has been solved by X-ray crystallography. The pictures below
are from Abrahams, J.P., Leslie, A.G., Lutter, R. and Walker, J.E.
(1994) Structure at 2.8 Å resolution of F1-ATPase from bovine
heart mitochondria
The ATP synthase operates through a mechanism in which
the three active sites undergo a change in binding affinity for the reactants
of the ATP-ase reaction, ATP, ADP and phosphate, as originally predicted by Paul Boyer. The change in affinity
accompanies a change in the position of the g-subunit relative to the a, b-ring, which involves a rotation of the one relative to the other. In the
direction of ATP synthesis, the rotation is driven by a flux of H+
down the proton gradient, through a coupling between the g-subunit, and the c-subunit of FO. This rotation has now been
demonstrated experimentally.
Respiratory control
The dependence of oxidative
phosphorylation on ADP reveals an important general feature of this process: Respiration
is tightly coupled to the synthesis of ATP. Not only is ATP synthesis
absolutely dependent on continued electron flow from substrates to oxygen, but
electron flow in normal mitochondria occurs only when ATP is being synthesized
as well. This regulatory phenomenon, called respiratory control, makes
biological sense, because it ensures that substrates will not be oxidized
wastefully. Instead, their utilization is controlled by the physiological need
for ATP.
In most aerobic cells the level of ATP exceeds that of
ADP by 4- to 10-fold. Respiration depends on ADP as a substrate for
phosphorylation. When ATP is consumed at high rates, accumulation of ADP
stimulates respiration, with concomitant activation of ATP resynthesis.
Conversely, in a relaxed and well-nourished cell, ATP accumulates at the
expense of ADP, and the depletion of ADP limits the rate of both electron
transport and its own phosphorylation to ATP. Thus, the energy-generating
capacity of the cell is closely attuned to its energy demands.
Experimentally, respiratory control
is demonstrated by following oxygen utilization in isolated mitochondria. In the
absence of added substrate or ADP, oxygen uptake, caused by oxidation of
endogenous substrates, is slow. Addition of an oxidizable substrate, such as
glutamate or malate, has but a small effect on the respiration rate. If ADP is
then added, however, oxygen uptake proceeds at an enhanced rate until all of
the added ADP has been converted to ATP, and then oxygen uptake returns to the
basal rate. This stimulation of respiration is stoichiometric; that is,
addition of twice as much ADP causes twice the amount of oxygen uptake at the
enhanced rate. If excess ADP is present instead of oxidizable substrate, the
addition of substrate in limiting amounts will stimulate oxygen uptake until
the substrate is exhausted.
Two mechanisms of the control
of respiration and ATP synthesis in mitochondria according to the utilization
of energy (ATP). The first mechanism of respiratory control is based on the
proton motive force Δp
across the inner mitochondrial membrane (grey). Activation of the ATP-synthase
(blue) by ADP, taken up via the ATP/ADP carrier (margenta), decreases Δp which in consequence stimulates the
three proton pumps of the respiratory chain (complexes I, III and IV). For
simplicity, only complex IV (cytochrome c
oxidase) and its substrate (cytochrome c)
are shown in green and red, respectively. The second mechanism of respiratory
control is based on the intramitochondrial ATP/ADP ratio. High ATP/ADP ratios
inhibit cytochrome c oxidase
activity allosterically. Uptake of ADP decreases the intramitochondrial ATP/ADP
ratio accompanied by exchange of bound ATP by ADP at the matrix domain of
subunit IV of cytochrome c
oxidase, with subsequent stimulation of respiration
Maintenance of respiratory control depends on the
structural integrity of the mitochondrion. Disruption of the organelle causes
electron transport to become uncoupled from ATP synthesis. Under these
conditions, oxygen uptake proceeds at high rates even in the absence of added
ADP. ATP synthesis is inhibited, even though electrons are being passed along
the respiratory chain and used to reduce O2 to water.
Uncoupling of respiration from phosphorylation can also
be achieved chemically. Chemical uncouplers such as DNP
or FCCP
act by dissipating the proton gradient. Addition of an uncoupler to
mitochondria stimulates oxygen utilization even in the absence of added ADP. No
phosphorylation occurs under these conditions because there is no ADP to be
phosphorylated.
The phenomenon of respiratory control
is the subject of today's studio
exercise. An oxygen electrode may be used to record [O2]
in a closed vessel (diagram p. 804). Electron transfer, e.g., from NADH to O2,
is monitored by recording the rate of disappearance of O2. At
right is an idealized representation of an oxygen electrode recording while
mitochondria respire in the presence of Pi, along with an electron
donor (e.g., succinate, or a substrate of a reaction that will generate NADH).
The dependence of respiration rate on availability of ADP, the substrate for
the ATP Synthase, is called respiratory control. The respiratory
control ratio is the ratio of slopes after and before ADP addition (b/a).
The P/O ratio is the moles of ADP added,
divided by the moles of O consumed (based on c) while phosphorylating
the added ADP.
Inhibitors of tissue respiration
Chemiosmosis can be disrupted by a variety of chemicals.
In oxidative phosphorylation, some of these inhibitors are quite infamous:
The
electron transport chain was determined by studying the effects of particular
inhibitors.
Rotenone
is a common insecticide that strongly inhibits the electron transport of
complex I.
Rotenone is a natural product obtained from the
roots of several species of plants. Tribes in certain parts of the world beat
the roots of trees along riverbanks to release rotenone into the water which
paralyzes fish and makes them easy prey.
Amytal is a barbiturate that inhibits the
electron transport of complex I. Demerol is painkiller that also inhibits
complex
Antibiotic. Induces apoptosis, which is not
prevented by the presence of Bcl-2. Inhibits mitochondrial electron transport
specifically between cytochromes b and c1. All three of these complex I
inhibitors block the oxidation of the Fe-S clusters of complex I.
2-Thenoyltrifluoroacetone
and carboxin specifically block electron transport in Complex II
Antimycin A is an antibiotic that inhibits
electron transfer in complex III by blocking the transfer of electrons between
Cyt bH and coenzyme Q bound at the QN site. Antibiotic. Induces apoptosis,
which is not prevented by the presence of Bcl-2. Inhibits mitochondrial
electron transport specifically between cytochromes b and c1.
Cyanide, azide and carbon monoxide all inhibit
electron transport in Complex IV. The all inhibit electron transfer by binding
tightly with the iron coordinated in Cyt a
This complex oxidizes cytochrome c and
also reduces O2 to H2O. Remember that
cytochromes have heme cofactors -- this is important in our discussion of
cyanide and azide. Cytochrome c is a soluble protein and also
is a mobile carrier. Other inhibitors of cytochrome c oxidase will not be
discussed here, but are important biologically, such as sulfide, formate, and
nitric oxide.
Azide and cyanide bind to the iron when the iron
is in the ferric state. Carbon Monoxide binds to the iron when it is in the ferrous
state. Cyanide and azide are potent inhibitors at this site which accounts for
there acute toxicity. Carbon monoxide is toxic due to its affinity for the heme
iron of hemoglobin. Animals carry many molecules of hemoglobin, therefore it
takes a large quantity of carbon monoxide to die from carbon monoxide
poisoning.
CO competes with oxygen for binding to the reduced form of
cytochrome c oxidase. Once bound to the cytochrome oxidase, oxygen cannot
attach, and electron transport is stopped. CO is a colorless, tasteless,
non-irritating toxic gas. When inhaled, the toxic gas enters the
bloodstream, depriving the heart and brain of the oxygen necessary to function
correctly. Sensing the body's need for more oxygen, the victim's heart rate
increases to pump more blood to the body's organs. If a person continues to
inhale CO, he or she faces the risk of breathing difficulty, cardiac trauma,
brain damage, coma and even death.
CN-, like azide, binds to the iron atom of oxidized
cytochrome, preventing binding of oxygen. Again, since cytochrome oxidase
is inhibited, oxygen metabolism is prevented and thus so is energy
generation. Cyanide has long been known as a poison, sometimes used in
warfare. As mentioned above, lethal doses cause death in 15 minutes.
They all cause similar toxic repercussions (with the exception of Carbon
Monoxide, which can bind to Hemoglobin causing your body to be unable to bind
oxygen properly and causing you to suffocate);
However, these three also cause chemical suffocation. They bind a protein in
our electron transport chain (cytochrome c oxidase). The electron transport
chain is our body's best way to create energy in the form of ATP. Without it,
we literally have next to no energy. The electron transport chain is the reason
we have pain in our muscles when we work out--if our body doesn't have oxygen
in those tissues and can't make energy, we make lactic acid, which causes our
muscles to hurt/burn. Anyway, cyanide, azide and carbon monoxide bind this
protein (cytochrome c oxidase) in the electron transport chain. Doing this
causes all of the electrons to stop transferring and no energy to be made. This
causes cell death. The first place to experience cell death from cyanide, azide
and carbon monoxide is your central nervous system--which is made up of your
brain and spinal cord.
So the first thing to die when someone dies of cyanide, azide or carbon
monoxide poisoning?
The coupling between electron transport and oxidative phosphorylation
depends on the impermeability of the inner mitochondrial membrane to
H+translocation. The only way for protons to go from the intermembrane space to
the matrix is through ATP synthase. Uncouplers uncouple electron transport from
oxidative phosphorylation. They collapse the chemiosmotic gradient by
dissipating protons across the inner mitochondrial membrane. All of the
uncouplers shown to the left, collapse the pH gradient by binding a proton on
the acidic side of the membrane, diffusing through the inner mitochondrial
membrane and releasing the proton on the membranes alkaline side.
Uncouplers of oxidative phosphorylation stimulate
the rate of electron flow but not
ATP synthesis.
(a)At relatively low levels of an uncoupling
agent, P/O ratios drop somewhat, but the cell can compensate for this by
increasing the rate of electron flow; ATP levels can be kept relatively normal.
At high levels of uncoupler, P/O ratios approach zero and the cell cannot
maintain ATP levels.
(b) As amounts of an uncoupler increase, the P/O
ratio decreases and the body struggles to make sufficient ATP by oxidizing more
fuel. The heat produced by this increased rate of oxidation raises the body
temperature. The P/O ratio is affected as noted in (a).
(c) Increased activity of the respiratory chain
in the presence of an uncoupler requires the degradation of additional energy
stores (glycogen and fat). By oxidizing more fuel in an attempt to produce the
same amount of ATP, the organism loses weight. If the P/O ratio nears zero, the
lack of ATP will be lethal
2,4-Dinitrophenol, dicumarol and carbonyl
cyanide-p-trifluorocarbonyl-cyanide methoxyphenyl hydrazone (FCCP) all have
hydrophobic character making them soluble in the bilipid membrane. All of these
decouplers also have dissociable protons allowing them to carry protons from
the intermembrane space to the matrix which collapses the pH gradient. The potential
energy of the proton gradient is lost as heat DNP is a chemical
uncoupler of electron transport and oxidative phosphorylation.
DNP
permeabilizes the inner mitochondrial membrane to protons, destroying the proton
gradient and, in doing so, uncouples the electron transport system from the
oxidative phosphorylation. In this situation, electrons continue to pass
through the electron transport system and reduce oxygen to water, but ATP is not
synthesized in the process. The compound, trifluorocarbonylcyanide
phenylhydrazone (FCCP), is also an
uncoupler.
The phenolic group of DNP is usually
dissociated at intracellular pH. However, a DNP molecule that approaches
the inner mitochondrial membrane from the outside becomes protonated (because
the pH is lower there). Protonation increases the hydrophobicity of DNP,
allowing it to diffuse into the membrane and, by mass action, to pass through.
Once inside, the higher pH of the matric deprotonates the phenolic hydroxyl
again. Thus, DNP has the effect of transporting H+ back
into the matrix, bypassing the F0 proton channel and thereby preventing ATP
synthesis.
The link between electron transport and ATP synthesis is below. (a) In
the presence of excess phosphate and substrate and intact mitochondria, oxygen
is consumed only when ADP is added. When all of the added ADP has been
converted into ATP, electron transport stops and oxygen consumption ceases. (b)
The addition of 2,4-dintrophenol uncouples electron transfer from ATP
synthesis. The oxygen is completely consumed in the absence of ADP. Endogenous
Uncouplers Enable Organisms to Generate Heat
.
The uncoupling of oxidative phosphorylation from
electron transport generates heat. Hibernating animals and newborne animals
(including human beings) contain brown adipose tissue. The adipose tissue is
brown due to the high mitochondria content of the tissue. An endogenous protein
called thermogenin uncouples ATP synthesis from electron transport by opening
up a passive proton channel (UCP-1) through the inner mitochondrial membrane.
The collapse of the pH gradient generates heat. An uncoupling
protein (also called thermogenin) is produced in brown adipose tissue
of newborn mammals and hibernating mammals .
This protein of the inner mitochondrial membrane
functions as a H+ carrier. The uncoupling protein
blocks development of a H+ electrochemical gradient, thereby
stimulating respiration. The free energy change associated with respiration is
dissipated as heat. This "non-shivering thermogenesis" is costly in
terms of respiratory energy unavailable for ATP synthesis, but it provides
valuable warming of the organism.
Valinomycin
combines with K ions to form a complex that passes through the inner
Valinomycin is a potent antibiotic which acts as a
potassium (K+) ionophore. Induces K+ conductivity in
cell membranes. Also active in vitro against Mycobacterium
Tuberculosis, and as an apoptosis inducer.
Valinomycin is obtained from the cells of several Streptomyces
strains, among which "S.
tsusimaensis" and S. fulvissimus.
It is a member of the group of natural neutral ionophores because it does not
have a residual charge. It consists of enantiomers D- and L-valine (Val),
D-hydroxyvaleric acid and L-lactic acid. Structures are alternately bound via
amide and ester bridges. Valinomycin is highly selective for potassium ions
over sodium ions within the cell membrane. It functions as a potassium-specific
transporter and facilitates the movement of potassium ions through lipid
membranes "down" an electrochemical potential gradient. The stability
constant K for the potassium-valinomycin complex is 106 and for the
sodium-valinomycin complex only 10. This difference is important for
maintaining the selectivity of valinomycin for the transport of potassium ions
(and not sodium ions) in biological systems.
Oligomycin
is a natural antibiotic isolated from Streptomyces diastatochromogenes which
inhibits mitochondrial H+-ATP synthase. It is primarily found to act
as an inhibitor of mitochondrial respiration and swelling. This
antibiotic is widely used as an inhibitor of oxidative phosphorylation.
Oligomycin inhibits the H+- ATP-synthase by binding to the
Oligomycin sensitivity-conferring protein (OSCP) at the F(o) subunits 6
and 9 which are found in the stalk of the F1F0-ATPase
complex. This binding blocks the proton conductance and inhibits the synthesis
of mitochondrial ATP.3-4 Because of its activity, it can also be
used to reduce the number of parameters (such as ER Ca2+
release, exocytotoxicity and apopotosis) which are affected by mitochondrial
depolarization.
Oligomycin, at high concentrations may also inhibit the plasma membrane
Na+-K+-ATPase. Interaction of Oligomycin with the Na+
occlusion site on the extracellular side of Na/K-ATPase, delays Na+
release to the extracellular side without inducing a conformational change.
Although Oligomycin stimulated Na+ binding to Na+/K+-ATPase,
it inhibited Na+/Na+ exchange, and did not affect
either Na+-dependent AD/-ATP exchange or K+-dependent
phosphatase activity.6-16
Proton Motive Force Drives Transport The primary purpose of the proton
gradient is togenerate ATP by oxidative phosphorylation. The potential energy
of the gradient can also be used for active transport. The inner mitochondrial
membrane is impermeable to charged molecules.
ATP-ADP Translocase There are two specific systems to transport ADP and
Pi into the mitochondrial matrix. A specific transport protein ATP-ADP
translocase enables ATP and ADP to transverse the inner mitochondrial membrane.
The transport of ADP in and ATP out are coupled. ADP only enters the matrix if
ATP exits or vice versa. ATP-ADP translocase has a single nucleotide binding
site which binds ADP and ATP with equal affinity. Due to the negative
electrostatic charge of the matrix, ATP-is bound on the N phase of the membrane
because it has greater negative charge than ADP.
Pentachlorophenol
(PCP) acts in a similar way to DNP. It was widely used
as a biocide, especially in pallet board manufacture as a fungicide, but is now
banned by the Biocidal Products Directive, because of its extreme toxicity and
environmental persistence.
DNP
PCP
Ways of
energy usage in the organism.
o
1. From the smallest, single-celled
organism to the biggest and most complex mammals--including people--all living
things require energy for life. It's easy enough to understand that we and
other animals eat. Things get a little more puzzling when we think about fungi,
which absorb their food as organic molecules, from the surrounding environment.
Where do those molecules come from? Furthermore, where does the food come from
that we humans convert to energy? At the most basic level, all energy traces
back to plants. Plants are the basis of all the world's food systems, and their
unique ability to make organic materials from sunlight--called
photosynthesis--is what sustains nearly every other life form on the planet.
o
The powerhouse of energy production
in all plants is called a chloroplast. More than a million of these handy
devices occur in every quarter-inch of a leaf. They contain the pigment called
chlorophyll that makes most leaves green--and drives photosynthesis. The
reaction isn't all that complicated, as far as chemical reactions go. The
chloroplasts take in carbon dioxide, sunlight and water. They release oxygen
and a bit less water than they took in. The conversion of carbon dioxide to
oxygen is one life-sustaining function that plants perform for Earth and all of
its life. But plants do something equally as important when they keep a third
product behind: glucose, the sugar that sustains the plants---and anything, in
turn, that eats the plants.
o
In cellular respiration, glucose is
broken down by the removal of its hydrogen atoms. That process releases energy
in the form of electrons, negatively charged particles that fuel all of a
cell's other work in later reactions. So, plants make the glucose and
everything down the line---from plant-eaters to the carnivores that eat
them---break the glucose down again, and use its energy. That's the simple
story. Of course, life is rarely so simple, and there are exceptions to every
rule. Every so often, a new discovery comes along about living things that use
a non-living substance other than sunlight to make energy--like ammonia, or
even sulfur. These less-common organisms can harness electrons from chemical
sources instead of the sun. More amazing life forms have the potential to be
discovered at any time, anywhere on our planet---or beyond.
o
Cytochrome P450
The active site of cytochrome P450
contains a heme
iron center. The iron is tethered to the P450 protein via a thiolate
ligand derived from a cysteine
residue. This cysteine and several flanking residues are highly conserved in
known CYPs and have the formal PROSITE
signature consensus pattern [FW] - [SGNH] - x - [GD] - {F} - [RKHPT] - {P} - C
- [LIVMFAP] - [GAD].[7]
Because of the vast variety of reactions catalyzed by CYPs, the activities and
properties of the many CYPs differ in many aspects.