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Carbohydrates
A carbohydrate is a biological molecule consisting of carbon (C), hydrogen (H) and oxygen (O)
atoms, usually with a hydrogen–oxygen atom ratio of 2:1 with the empirical formula Cm(H2O)n
(where m could be different from n). Carbohydrates are technically hydrates of carbon
structurally it is more accurate to view them as polyhydroxy aldehydes and ketones. The
saccharides are divided into four chemical groups: monosaccharides, disaccharides,
oligosaccharides, and polysaccharides. In general, the monosaccharides and disaccharides, which
are smaller (lower molecular weight) carbohydrates, are commonly referred to as sugars.[6] The
word saccharide comes from the Greek word (sákkharon), meaning "sugar". While the scientific
nomenclature of carbohydrates is complex, the names of the monosaccharides and disaccharides
very often end in the suffix -ose.
Carbohydrates perform numerous roles in living organisms. Polysaccharides serve for the storage
of energy (e.g. starch and glycogen) and as structural components (e.g. cellulose in plants and
chitin in arthropods). The 5-carbon monosaccharide ribose is an important component of
coenzymes (e.g. ATP, FAD and NAD) and the backbone of the genetic molecule known as
RNA. The related deoxyribose is a component of DNA. Saccharides and their derivatives include
many other important biomolecules that play key roles in the immune system, fertilization,
preventing pathogenesis, blood clotting, and development.
Structure
Formerly the name "carbohydrate" was used in chemistry for any compound with the formula Cm
(H2O)n. Following this definition, some chemists considered formaldehyde (CH2O) to be the
simplest carbohydrate, while others claimed that title for glycolaldehyde.
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Natural saccharides are generally built of simple carbohydrates called monosaccharides with
general formula (CH2O)n where n is three or more. A typical monosaccharide has the structure
H–(CHOH)x(C=O)–(CHOH)y–H, that is, an aldehyde or ketone with many hydroxyl groups
added, usually one on each carbon atom that is not part of the aldehyde or ketone functional
group. Examples of monosaccharides are glucose, fructose, and glyceraldehydes. However, some
biological substances commonly called "monosaccharides" do not conform to this formula (e.g.
uronic acids and deoxy-sugars such as fucose) and there are many chemicals that do conform to
this formula but are not considered to be monosaccharides (e.g. formaldehyde CH2O and inositol
(CH2O)6).
The open-chain form of a monosaccharide often coexists with a closed ring form where the
aldehyde/ketone carbonyl group carbon (C=O) and hydroxyl group (–OH) react forming a
hemiacetal with a new C–O–C bridge.
Monosaccharides can be linked together into what are called polysaccharides (or
oligosaccharides) in a large variety of ways. Many carbohydrates contain one or more modified
monosaccharide units that have had one or more groups replaced or removed. For example,
deoxyribose, a component of DNA, is a modified version of ribose; chitin is composed of
repeating units of N-acetyl glucosamine, a nitrogen-containing form of glucose.
Class
Subgroup
Components
Sugars (1–2)
Monosaccharides
Glucose, galactose, fructose, xylose
Disaccharides
Sucrose, lactose, maltose, trehalose
Polyols
Sorbitol, mannitol
Oligosaccharides
Malto-oligosaccharides
Maltodextrins
(3–9)
Other oligosaccharides
Raffinose,
stachyose,
fructo-
oligosaccharides
Polysaccharides
Starch
Amylose, amylopectin, modified starches
(>9)
Non-starch
Cellulose,
polysaccharides
hydrocolloids
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hemicellulose,
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pectins,
Monosaccharide
D-glucose is an aldohexose with the formula (C·H2O)6. The red atoms highlight the aldehyde
group and the blue atoms highlight the asymmetric center furthest from the aldehyde; because
this -OH is on the right of the Fischer projection, this is a D sugar.
Monosaccharides are the simplest carbohydrates in that they cannot be hydrolyzed to smaller
carbohydrates. They are aldehydes or ketones with two or more hydroxyl groups. The general
chemical formula of an unmodified monosaccharide is (C•H2O)n, literally a "carbon hydrate".
Monosaccharides are important fuel molecules as well as building blocks for nucleic acids. The
smallest monosaccharides, for which n=3, are dihydroxyacetone and D- and L-glyceraldehydes.
Classification of monosaccharides
The α and β anomers of glucose. Note the position of the hydroxyl group (red or green) on the
anomeric carbon relative to the CH2OH group bound to carbon 5: they either have identical
absolute configurations (R,R or S,S) (α), or opposite absolute configurations (R,S or S,R) (β).
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Monosaccharides are classified according to three different characteristics: the placement of its
carbonyl group, the number of carbon atoms it contains, and its chiral handedness. If the
carbonyl group is an aldehyde, the monosaccharide is an aldose; if the carbonyl group is a
ketone, the monosaccharide is a ketose. Monosaccharides with three carbon atoms are called
trioses, those with four are called tetroses, five are called pentoses, six are hexoses, and so on.
These two systems of classification are often combined. For example, glucose is an aldohexose
(a six-carbon aldehyde), ribose is an aldopentose (a five-carbon aldehyde), and fructose is a
ketohexose (a six-carbon ketone).
Each carbon atom bearing a hydroxyl group (-OH), with the exception of the first and last
carbons, are asymmetric, making them stereo centers with two possible configurations each (R or
S). Because of this asymmetry, a number of isomers may exist for any given monosaccharide
formula. Using Le Bel-van't Hoff rule, the aldohexose D-glucose, for example, has the formula
(C·H2O)6, of which four of its six carbons atoms are stereogenic, making D-glucose one of 24=16
possible stereoisomers. In the case of glyceraldehydes, an aldotriose, there is one pair of possible
stereoisomers, which are enantiomers and epimers. 1, 3-dihydroxyacetone, the ketose
corresponding to the aldose glyceraldehydes, is a symmetric molecule with no stereo centers.
The assignment of D or L is made according to the orientation of the asymmetric carbon furthest
from the carbonyl group: in a standard Fischer projection if the hydroxyl group is on the right the
molecule is a D sugar, otherwise it is an L sugar. The "D-" and "L-" prefixes should not be
confused with "d-" or "l-", which indicate the direction that the sugar rotates plane polarized
light. This usage of "d-" and "l-" is no longer followed in carbohydrate chemistry.[16]
Ring-straight chain isomerism
Glucose can exist in both a straight-chain and ring form.
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The aldehyde or ketone group of a straight-chain monosaccharide will react reversibly with a
hydroxyl group on a different carbon atom to form a hemiacetal or hemiketal, forming a
heterocyclic ring with an oxygen bridge between two carbon atoms. Rings with five and six
atoms are called furanose and pyranose forms, respectively, and exist in equilibrium with the
straight-chain form.
During the conversion from straight-chain form to the cyclic form, the carbon atom containing
the carbonyl oxygen, called the anomeric carbon, becomes a stereogenic center with two possible
configurations: The oxygen atom may take a position either above or below the plane of the ring.
The resulting possible pair of stereoisomers is called anomers. In the α anomer, the -OH
substituent on the anomeric carbon rests on the opposite side (trans) of the ring from the CH2OH
side branch. The alternative form, in which the CH2OH substituent and the anomeric hydroxyl
are on the same side (cis) of the plane of the ring, is called the β anomer.
Functions of monosacchrides
Monosaccharides are the major source of fuel for metabolism, being used both as an energy
source (glucose being the most important in nature) and in biosynthesis. When monosaccharides
are not immediately needed by many cells they are often converted to more space-efficient
forms, often polysaccharides. In many animals, including humans, this storage form is glycogen,
especially in liver and muscle cells. In plants, starch is used for the same purpose. The most
abundant carbohydrate, cellulose, is a structural component of the cell wall of plants and many
forms of algae. Ribose is a component of RNA. Deoxyribose is a component of DNA. Lyxose is
a component of lyxoflavin found in the human heart.[18] Ribulose and xylulose occur in the
pentose phosphate pathway. Galactose, a component of milk sugar lactose, is found in
galactolipids in plant cell membranes and in glycoproteins in many tissues. Mannose occurs in
human metabolism, especially in the glycosylation of certain proteins. Fructose, or fruit sugar, is
found in many plants and in humans, it is metabolized in the liver, absorbed directly into the
intestines during digestion, and found in semen. Trehalose, a major sugar of insects, is rapidly
hydrolyzed into two glucose molecules to support continuous flight.
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Disaccharides
Sucrose, also known as table sugar, is a common disaccharide. It is composed of two
monosaccharides: D-glucose (left) and D-fructose (right).
Two joined monosaccharides are called a disaccharide and these are the simplest
polysaccharides. Examples include sucrose and lactose. They are composed of two
monosaccharide units bound together by a covalent bond known as a glycosidic linkage formed
via a dehydration reaction, resulting in the loss of a hydrogen atom from one monosaccharide
and a hydroxyl group from the other. The formula of unmodified disaccharides is C12H22O11.
Sucrose is the most abundant disaccharide, and the main form in which carbohydrates are
transported in plants. It is composed of one D-glucose molecule and one D-fructose molecule.
The systematic name for sucrose, O-α-D-glucopyranosyl-(1→2)-D-fructofuranoside, indicates
four things:

Its monosaccharides: glucose and fructose

Their ring types: glucose is a pyranose and fructose is a furanose

How they are linked together: the oxygen on carbon number 1 (C1) of α-D-glucose is
linked to the C2 of D-fructose.

The -oside suffix indicates that the anomeric carbon of both monosaccharides participates
in the glycosidic bond.
Lactose, a disaccharide composed of one D-galactose molecule and one D-glucose molecule,
occurs naturally in mammalian milk. The systematic name for lactose is O-β-Dgalactopyranosyl-(1→4)-D-glucopyranose. Other notable disaccharides include maltose (two Dglucoses linked α-1,4) and cellulobiose (two D-glucoses linked β-1,4). Disaccharides can be
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classified into two types: reducing and non-reducing disaccharides. If the functional group is
present in bonding with another sugar unit, it is called a reducing disaccharide or biose.
Polysaccharide
Polysaccharides are polymeric carbohydrate molecules composed of long chains of
monosaccharide units bound together by glycosidic linkages and on hydrolysis give the
constituent monosaccharides or oligosaccharides. They range in structure from linear to highly
branched. Examples include storage polysaccharides such as starch and glycogen, and structural
polysaccharides such as cellulose and chitin.
Polysaccharides are often quite heterogeneous, containing slight modifications of the repeating
unit. Depending on the structure, these macromolecules can have distinct properties from their
monosaccharide building blocks. They may be amorphous or even insoluble in water.[1] When all
the monosaccharides in a polysaccharide are the same type, the polysaccharide is called a
homopolysaccharide or homoglycan, but when more than one type of monosaccharide is present
they are called heteropolysaccharides or heteroglycans.
Natural saccharides are generally of simple carbohydrates called monosaccharides with general
formula (CH2O)n where n is three or more. Examples of monosaccharides are glucose, fructose,
and glyceraldehyde.[4] Polysaccharides, meanwhile, have a general formula of Cx(H2O)y where x
is usually a large number between 200 and 2500. When the repeating units in the polymer
backbone are six-carbon monosaccharides, as is often the case, the general formula simplifies to
(C6H10O5)n, where typically 40≤n≤3000.
As a rule of thumb, polysaccharides contain more than ten monosaccharide units, whereas
oligosaccharides contain three through ten monosaccharide units; but the precise cutoff varies
somewhat according to convention. Polysaccharides are an important class of biological
polymers. Their function in living organisms is usually either structure- or storage-related. Starch
(a polymer of glucose) is used as a storage polysaccharide in plants, being found in the form of
both amylose and the branched amylopectin. In animals, the structurally similar glucose polymer
is the more densely branched glycogen, sometimes called 'animal starch'. Glycogen's properties
allow it to be metabolized more quickly, which suits the active lives of moving animals.
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Cellulose and chitin are examples of structural polysaccharides. Cellulose is used in the cell
walls of plants and other organisms, and is said to be the most abundant organic molecule on
Earth. It has many uses such as a significant role in the paper and textile industries, and is used
as a feedstock for the production of rayon (via the viscose process), cellulose acetate, celluloid,
and nitrocellulose. Chitin has a similar structure, but has nitrogen-containing side branches,
increasing its strength. It is found in arthropod exoskeletons and in the cell walls of some fungi.
It also has multiple uses, including surgical threads. Polysaccharides also include callose or
laminarin, chrysolaminarin, xylan, arabinoxylan, mannan, fucoidan and galactomannan.
Function
Nutrition polysaccharides are common sources of energy. Many organisms can easily break
down starches into glucose; however, most organisms cannot metabolize cellulose or other
polysaccharides like chitin and arabinoxylans. These carbohydrate types can be metabolized by
some bacteria and protists. Ruminants and termites, for example, use microorganisms to process
cellulose. Even though these complex carbohydrates are not very digestible, they provide
important dietary elements for humans. Called dietary fiber, these carbohydrates enhance
digestion among other benefits. The main action of dietary fiber is to change the nature of the
contents of the gastrointestinal tract, and to change how other nutrients and chemicals are
absorbed. Soluble fiber binds to bile acids in the small intestine, making them less likely to enter
the body; this in turn lowers cholesterol levels in the blood.[8] Soluble fiber also attenuates the
absorption of sugar, reduces sugar response after eating, normalizes blood lipid levels and, once
fermented in the colon, produces short-chain fatty acids as byproducts with wide-ranging
physiological activities. Although insoluble fiber is associated with reduced diabetes risk, the
mechanism by which this occurs is unknown.
Storage polysaccharides
Starch
Starch is a glucose polymer in which glucopyranose units are bonded by alpha-linkages. It is
made up of a mixture of amylose (15–20%) and amylopectin (80–85%). Amylose consists of a
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linear chain of several hundred glucose molecules and Amylopectin is a branched molecule
made of several thousand glucose units (every chain of 24–30 glucose units is one unit of
Amylopectin). Starches are insoluble in water. They can be digested which can break the alphalinkages (glycosidic bonds). Both humans and animals have amylases, so they can digest
starches. Potato, rice, wheat, and maize are major sources of starch in the human diet. The
formations of starches are the ways that plants store glucose. .
Glycogen
Glycogen serves as the secondary long-term energy storage in animal and fungal cells, with the
primary energy stores being held in adipose tissue. Glycogen is made primarily by the liver and
the muscles, but can also be made by glycogenesis within the brain and stomach.
Glycogen is the analogue of starch, a glucose polymer in plants, and is sometimes referred to as
animal starch, having a similar structure to amylopectin but more extensively branched and
compact than starch. Glycogen is a polymer of α(1→4) glycosidic bonds linked, with α(1→6)linked branches. Glycogen is found in the form of granules in the cytosol/cytoplasm in many cell
types, and plays an important role in the glucose cycle. Glycogen forms an energy reserve that
can be quickly mobilized to meet a sudden need for glucose, but one that is less compact and
more immediately available as an energy reserve than triglycerides (lipids).
In the liver hepatocytes, glycogen can compose up to eight percent (100–120 g in an adult) of the
fresh weight soon after a meal.[14] Only the glycogen stored in the liver can be made accessible to
other organs. In the muscles, glycogen is found in a low concentration of one to two percent of
the muscle mass. The amount of glycogen stored in the body—especially within the muscles,
liver, and red blood cells varies with physical activity, basal metabolic rate, and eating habits
such as intermittent fasting. Small amounts of glycogen are found in the kidneys, and even
smaller amounts in certain glial cells in the brain and white blood cells. The uterus also stores
glycogen during pregnancy, to nourish the embryo.[14]
Glycogen is composed of a branched chain of glucose residues. It is stored in liver and muscles.

It is an energy reserve for animals.
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
It is the chief form of carbohydrate stored in animal body.

It is insoluble in water. It turns brown-red when mixed with iodine.

It also yields glucose on hydrolysis.
Structural polysaccharides
Cellulose
The structural component of plants are formed primarily from cellulose. Wood is largely
cellulose and lignin, while paper and cotton are nearly pure cellulose. Cellulose is a polymer
made with repeated glucose units bonded together by beta-linkages. Humans and many animals
lack an enzyme to break the beta-linkages, so they do not digest cellulose. Certain animals such
as termites can digest cellulose, because bacteria possessing the enzyme are present in their gut.
Cellulose is insoluble in water. It does not change color when mixed with iodine. On hydrolysis,
it yields glucose. It is the most abundant carbohydrate in nature.
Chitin
Chitin is one of many naturally occurring polymers. It forms a structural component of many
animals, such as exoskeletons. Over time it is bio-degradable in the natural environment. Its
breakdown may be catalyzed by enzymes called chitinases, secreted by microorganisms such as
bacteria and fungi, and produced by some plants. Some of these microorganisms have receptors
to simple sugars from the decomposition of chitin. If chitin is detected, they then produce
enzymes to digest it by cleaving the glycosidic bonds in order to convert it to simple sugars and
ammonia.
Pectins
Pectins are a family of complex polysaccharides that contain 1,4-linked α-D-galactosyl uronic
acid residues. They are present in most primary cell walls and in the non-woody parts of
terrestrial plants.
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Glycolysis
Glycolysis is a metabolic pathway that converts glucose C6H12O6, into pyruvate, and the free
energy released in this process is used to form the high-energy compounds ATP (adenosine
triphosphate) and NADH (reduced nicotinamide adenine dinucleotide).
Glycolysis occurs in most organisms in the cytosol of the cell. The entire glycolysis pathway can
be separated into two phases:
1. The Preparatory Phase – in which ATP is consumed and is hence also known as the
investment phase
2. The Pay Off Phase – in which ATP is produced.
Preparatory phase
The first five steps are regarded as the preparatory (or investment) phase, since they consume
energy to convert the glucose into two three-carbon sugar phosphates (G3P).
The first step in glycolysis is phosphorylation of glucose by a family of enzymes called
hexokinases to form glucose 6-phosphate (G6P). This reaction consumes ATP, but it acts to keep
the glucose concentration low, promoting continuous transport of glucose into the cell through
the plasma membrane transporters. In addition, it blocks the glucose from leaking out – the cell
lacks transporters for G6P, and free diffusion out of the cell is prevented due to the charged
nature of G6P. Glucose may alternatively be formed from the phosphorolysis or hydrolysis of
intracellular starch or glycogen.
In animals, an isozyme of hexokinase called glucokinase is also used in the liver, which has a
much lower affinity for glucose (Km in the vicinity of normal glycemia), and differs in regulatory
properties. The different substrate affinity and alternate regulation of this enzyme are a reflection
of the role of the liver in maintaining blood sugar levels.
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A second phosphorylation reaction follows the isomerization step. Fructose 6-phosphate is
phosphorylated by ATP to fructose 1,6-bisphosphate (F-1,6-BP). The prefix bis- in bisphosphate
means that two separate monophosphate groups are present, whereas the prefix di- in
diphosphate (as in adenosine diphosphate) means that two phosphate groups are present and are
connected by an anhydride bond.
This reaction is catalyzed by phosphofructokinase (PFK), an allosteric enzyme that sets the pace
of glycolysis (Section 16.2.1). As we will learn, this enzyme plays a central role in the
integration of much of metabolism.
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The Six-Carbon Sugar Is Cleaved into Two Three-Carbon Fragments by Aldolase
The second stage of glycolysis begins with the splitting of fructose 1,6-bisphosphate into
glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). The products of
the remaining steps in glycolysis consist of three-carbon units rather than six-carbon units.
Stage 2 of glycolysis. Two three-carbon fragments are produced from one six-carbon sugar.
This reaction is catalyzed by aldolase. This enzyme derives its name from the nature of the
reverse reaction, an aldol condensation. The reaction catalyzed by aldolase is readily reversible
under intracellular conditions.
Triose phosphate isomerase Salvages a Three-Carbon Fragment
Glyceraldehyde 3-phosphate is on the direct pathway of glycolysis, whereas dihydroxyacetone
phosphate is not. Unless a means exists to convert dihydroxyacetone phosphate into
glyceraldehyde 3-phosphate, a three-carbon fragment useful for generating ATP will be lost.
These compounds are isomers that can be readily interconverted: dihydroxyacetone phosphate is
a ketose, whereas glyceraldehyde 3-phosphate is an aldose. The isomerization of these threecarbon phosphorylated sugars is catalyzed by triose phosphate isomerase. This reaction is rapid
and reversible.
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Energy Transformation: Phosphorylation Is Coupled to the Oxidation of Glyceraldehyde
3-phosphate by a Thioester Intermediate
The preceding steps in glycolysis have transformed one molecule of glucose into two molecules
of glyceraldehyde 3-phosphate, but no energy has yet been extracted. On the contrary, thus far
two molecules of ATP have been invested. We come now to a series of steps that harvest some
of the energy contained in glyceraldehyde 3-phosphate. The initial reaction in this sequence is
the conversion of glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate (1,3-BPG), a
reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase (Figure 16.7).
Stage 3 of Glycolysis. The oxidation of three-carbon fragments yields ATP.
1,3-Bisphosphoglycerate is an acyl phosphate. Such compounds have a high phosphoryl-transfer
potential; one of its phosphoryl groups is transferred to ADP in the next step in glycolysis. The
reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase is really the sum of two
processes: the oxidation of the aldehyde to a carboxylic acid by NAD+ and the joining of the
carboxylic acid and orthophosphate to form the acyl-phosphate product.
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The Formation of ATP from 1,3-Bisphosphoglycerate
The final stage in glycolysis is the generation of ATP from the phosphorylated three-carbon
metabolites of glucose. Phosphoglycerate kinase catalyzes the transfer of the phosphoryl group
from the acyl phosphate of 1,3-bisphosphoglycerate to ADP. ATP and 3-phosphoglycerate are
the products.
The formation of ATP in this manner is referred to as substrate-level phosphorylation because
the phosphate donor, 1,3-BPG, is a substrate with high phosphoryl-transfer potential. We will
contrast this manner of ATP formation with that in which ATP is formed from ionic gradients in
Chapters 18 and 19.
Thus, the outcomes of the reactions catalyzed by glyceraldehyde 3-phosphate dehydrogenase and
phosphoglycerate kinase are:
1. Glyceraldehyde 3-phosphate, an aldehyde, is oxidized to 3-phosphoglycerate, a carboxylic
acid.
2. NAD+ is concomitantly reduced to NADH.
3. ATP is formed from Pi and ADP at the expense of carbon oxidation energy.
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The Generation of Additional ATP and the Formation of Pyruvate
In the remaining steps of glycolysis, 3-phosphoglycerate is converted into pyruvate with the
concomitant conversion of ADP into ATP.
The first reaction is a rearrangement. The position of the phosphoryl group shifts in the
conversion of 3-phosphoglycerate into 2-phosphoglycerate, a reaction catalyzed by
phosphoglycerate mutase. In general, a mutase is an enzyme that catalyzes the intramolecular
shift of a chemical group, such as a phosphoryl group. The phosphoglycerate mutase reaction has
an interesting mechanism: the phosphoryl group is not simply moved from one carbon to
another. This enzyme requires catalytic amounts of 2,3-bisphosphoglycerate to maintain an
active-site histidine residue in a phosphorylated form.
The sum of these reactions yields the mutase reaction:
Examination of the first partial reaction reveals that the mutase functions as a phosphatase—it
converts 2,3-bisphosphoglycerate into 2-phosphoglycerate. However, the phosphoryl group
remains linked to the enzyme. This phosphoryl group is then transferred to 3-phosphoglycerate
to reform 2,3-bisphosphoglycerate.
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In the next reaction, an enol is formed by the dehydration of 2-phosphoglycerate. Enolase
catalyzes the formation of phosphoenolpyruvate (PEP). This dehydration markedly elevates the
transfer potential of the phosphoryl group. An enol phosphate has a high phosphoryl-transfer
potential, whereas the phosphate ester, such as 2-phosphoglycerate, of an ordinary alcohol has a
low one. The ΔG°´ of the hydrolysis of a phosphate ester of an ordinary alcohol is -3 kcal mol-1
(- 13 kJ mol-1), whereas that of phosphoenolpyruvate is -14.8 kcal mol-1 (- 62 kJ mol-1). Why
does phosphoenolpyruvate have such a high phosphoryl-transfer potential? The phosphoryl
group traps the molecule in its unstable enol form. When the phosphoryl group has been donated
to ATP, the enol undergoes a conversion into the more stable ketone—namely, pyruvate.
Thus, the high phosphoryl-transfer potential of phosphoenolpyruvate arises primarily from the
large driving force of the subsequent enol-ketone conversion. Hence, pyruvate is formed, and
ATP is generated concomitantly. The virtually irreversible transfer of a phosphoryl group from
phosphoenolpyruvate to ADP is catalyzed by pyruvate kinase. Because the molecules of ATP
used in forming fructose 1,6-bisphosphate have already been regenerated, the two molecules of
ATP generated from phosphoenolpyruvate are “profit.”
Energy Yield in the Conversion of Glucose into Pyruvate
The net reaction in the transformation of glucose into pyruvate is:
Thus, two molecules of ATP are generated in the conversion of glucose into two molecules of
pyruvate.
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Regulation
3. Glycolysis is regulated by slowing down or speeding up certain steps in the pathway by
inhibiting or activating the enzymes that are involved. The steps that are regulated may
be determined by calculating the change in free energy, ΔG, for each step.
4. When ΔG is negative, a reaction proceeds spontaneously in the forward direction only
and is considered irreversible. When ΔG is positive, the reaction is non-spontaneous and
will not proceed in the forward direction unless coupled with an energetically favorable
reaction. When ΔG is zero, the reaction is at equilibrium, can proceed in either directions
and is considered reversible.
5. If a step is at equilibrium (ΔG is zero), the enzyme catalyzing the reaction will balance
the products and reactants and cannot confer directionality to the pathway. These steps
(and associated enzymes) are considered unregulated. If a step is not at equilibrium, but
spontaneous (ΔG is negative), the enzyme catalyzing the reaction is not balancing the
products and reactants and is considered to be regulated. A common mechanism of
regulating enzymes is allosteric control.
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Introduction
Overview of the citric acid cycle
In eukaryotes, the citric acid cycle takes place in the matrix of the mitochondria, just like the
conversion of pyruvate to acetyl CoA. In prokaryotes, these steps both take place in the
cytoplasm. The citric acid cycle is a closed loop; the last part of the pathway reforms the
molecule used in the first step. The cycle includes eight major steps.
First, acetyl CoA combines with oxaloacetate, a four-carbon molecule, losing the CoA group and
forming the six-carbon molecule citrate. After citrate undergoes a rearrangement step, it
undergoes an oxidation reaction, transferring electrons to NAD+ to form NADH and releasing a
molecule of carbon dioxide. The five-carbon molecule left behind then undergoes a second,
similar reaction, transferring electrons to NAD+ to form NADH and releasing a carbon dioxide
molecule. The four-carbon molecule remaining then undergoes a series of transformations, in the
course of which GDP and inorganic phosphate are converted into GTP—or, in some organisms,
ADP and inorganic phosphate are converted into ATP—an FAD molecule is reduced to FADH2,
and another NAD+ is reduced to NADH. At the end of this series of reactions, the four-carbon
starting molecule, oxaloacetate, is regenerated, allowing the cycle to begin again.
In the first step of the cycle, acetyl CoA combines with a four-carbon acceptor molecule,
oxaloacetate, to form a six-carbon molecule called citrate. After a quick rearrangement, this sixcarbon molecule releases two of its carbons as carbon dioxide molecules in a pair of similar
reactions, producing a molecule of NADH. The enzymes that catalyze these reactions are key
regulators of the citric acid cycle, speeding it up or slowing it down based on the cell’s energy
needs.
The remaining four-carbon molecule undergoes a series of additional reactions, first making an
ATP molecule or, in some cells, a similar molecule called GTPthen reducing the electron carrier
FAD to FADH2 and finally generating another NADH. This set of reactions regenerates the
starting molecule, oxaloacetate, so the cycle can repeat.
Overall, one turn of the citric acid cycle releases two carbon dioxide molecules and produces
three NADH, one FADH2 and one ATP or GTP. The citric acid cycle goes around twice for each
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molecule of glucose that enters cellular respiration because there are two pyruvates—and thus,
two acetyl CoA made per glucose.
Steps of the citric acid cycle
Step 1. In the first step of the citric acid cycle, acetyl CoA joins with a four-carbon molecule,
oxaloacetate, releasing the CoA group and forming a six-carbon molecule called citrate.
Step 2. In the second step, citrate is converted into its isomer, isocitrate. This is actually a twostep process, involving first the removal and then the addition of a water molecule, which is why
the citric acid cycle is sometimes described as having nine steps—rather than the eight.
Step 3. In the third step, isocitrate is oxidized and releases a molecule of carbon dioxide, leaving
behind a five-carbon molecule—α-ketoglutarate. During this step, NAD is reduced to form
NADH. The enzyme catalyzing this step, isocitrate dehydrogenase, is important in regulating
the speed of the citric acid cycle.
Step 4. The fourth step is similar to the third. In this case, it’s α-ketoglutarate that’s oxidized,
reducing NAD to NADH and releasing a molecule of carbon dioxide in the process. The
remaining four-carbon molecule picks up Coenzyme A, forming the unstable compound succinyl
CoA. The enzyme catalyzing this step, α-ketoglutarate dehydrogenase, is also important in
regulation of the citric acid cycle.
Step 1. Acetyl CoA combines with oxaloacetate in a reaction catalyzed by citrate synthase. This
reaction also takes a water molecule as a reactant, and it releases a SH-CoA molecule as a
product.
Step 2. Citrate is converted into isocitrate in a reaction catalyzed by aconitase.
Step 3. Isocitrate is converted into α-ketoglutarate in a reaction catalyzed by isocitrate
dehydrogenase. An NAD+ molecule is reduced to NADH + H+ in this reaction, and a carbon
dioxide molecule is released as a product.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
Step 4. α-ketoglutarate is converted to succinyl CoA in a reaction catalyzed by α-ketoglutarate
dehydrogenase. An NAD+ molecule is reduced to NADH + H+ in this reaction, which also takes
a SH-CoA molecule as reactant. A carbon dioxide molecule is released as a product.
Step 5. Succinyl CoA is converted to succinate in a reaction catalyzed by the enzyme succinylCoA synthetase. This reaction converts inorganic phosphate, Pi, and GDP to GTP and also
releases a SH-CoA group.
Step 6. Succinate is converted to fumarate in a reaction catalyzed by succinate dehydrogenase.
FAD is reduced to FADH2 in this reaction.
Step 7. Fumarate is converted to malate in a reaction catalyzed by the enzyme fumarase. This
reaction requires a water molecule as a reactant.
Step 8. Malate is converted to oxaloacetate in a reaction catalyzed by malate dehydrogenase. This
reaction reduces an NAD+ molecule to NADH + H+.
Step 5. In step five, the CoA of succinyl CoA A is replaced by a phosphate group, which is then
transferred to ADP to make ATP. In some cells, GDP guanine diphosphate—is used instead of
ADP forming GTP guanine triphosphate—as a product. The four-carbon molecule produced in
this step is called succinate.
Step 6. In step six, succinate is oxidized, forming another four-carbon molecule called fumarate.
In this reaction, two hydrogen atoms with their electrons are transferred to FAD producing
FADH2. The enzyme that carries out this step is embedded in the inner membrane of the
mitochondrion, so FADH2 can transfer its electrons directly into the electron transport chain.
Step 7. In step seven, water is added to the four-carbon molecule fumarate, converting it into
another four-carbon molecule called malate.
Step 8. In the last step of the citric acid cycle, oxaloacetate—the starting four-carbon
compound—is regenerated by oxidation of malate. Another molecule of NAD is reduced to
NADH in the process.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
Products of the citric acid cycle
In a single turn of the cycle,

two carbons enter from acetyl CoA and two molecules of carbon dioxide are released;

three molecules of NADH and one molecule of FADH2 are generated; and

one molecule of ATP or GTP is produced.
These figures are for one turn of the cycle, corresponding to one molecule of acetyl CoA. Each
glucose produces two acetyl CoA molecules, so we need to multiply these numbers by 2 if we
want the per-glucose yield.
Two carbons—from acetyl CoA enter the citric acid cycle in each turn, and two carbon dioxide
molecules are released. However, the carbon dioxide molecules don’t actually contain carbon
atoms from the acetyl CoA that just entered the cycle. Instead, the carbons from acetyl CoA are
initially incorporated into the intermediates of the cycle and are released as carbon dioxide only
during later turns. After enough turns, all the carbon atoms from the acetyl group of acetyl CoA
will be released as carbon dioxide.
Regulation
The regulation of the TCA cycle is largely determined by product inhibition and substrate
availability. If the cycle were permitted to run unchecked, large amounts of metabolic energy
could be wasted in overproduction of reduced coenzyme such as NADH and ATP. The major
eventual substrate of the cycle is ADP which gets converted to ATP. A reduced amount of ADP
causes accumulation of precursor NADH which in turn can inhibit a number of enzymes.
NADH, a product of all dehydrogenases in the TCA cycle with the exception of succinate
dehydrogenase, inhibits pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate
dehydrogenase, and also citrate synthase. Acetyl-coA inhibits pyruvate dehydrogenase, while
succinyl-CoA inhibits alpha-ketoglutarate dehydrogenase and citrate synthase. When tested in
vitro with TCA enzymes, ATP inhibits citrate synthase and α-ketoglutarate dehydrogenase;
however, ATP levels do not change more than 10% in vivo between rest and vigorous exercise.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
There is no known allosteric mechanism that can account for large changes in reaction rate from
an allosteric effector whose concentration changes less than 10%.
Calcium is used as a regulator. Mitochondrial matrix calcium levels can reach the tens of
micromolar levels during cellular activation.[27] It activates pyruvate dehydrogenase phosphatase
which in turn activates the pyruvate dehydrogenase complex. Calcium also activates isocitrate
dehydrogenase and α-ketoglutarate dehydrogenase.[28] This increases the reaction rate of many of
the steps in the cycle, and therefore increases flux throughout the pathway.
Citrate is used for feedback inhibition, as it inhibits phosphofructokinase, an enzyme involved in
glycolysis that catalyses formation of fructose 1,6-bisphosphate,a precursor of pyruvate. This
prevents a constant high rate of flux when there is an accumulation of citrate and a decrease in
substrate for the enzyme.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
Pentose phosphate pathway
The pentose phosphate pathway (also called the phosphogluconate pathway and the hexose
monophosphate shunt) is a metabolic pathway parallel to glycolysis that generates NADPH and
pentoses (5-carbon sugars) as well as Ribose 5-phosphate, a precursor for the synthesis of
nucleotides. While it does involve oxidation of glucose, its primary role is anabolic rather than
catabolic.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
There are two distinct phases in the pathway. The first is the oxidative phase, in which NADPH
is generated, and the second is the non-oxidative synthesis of 5-carbon sugars. For most
organisms, the pentose phosphate pathway takes place in the cytosol; in plants, most steps take
place in plastids.[1]
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
Oxidative phase
In this phase, two molecules of NADP+ are reduced to NADPH, utilizing the energy from the
conversion of glucose-6-phosphate into ribulose 5-phosphate.
Oxidative phase of pentose phosphate pathway
Glucose-6-phosphate (1),
6-phosphoglucono-δ-lactone (2),
6-phosphogluconate (3),
ribulose 5-phosphate (4)
The entire set of reactions can be summarized as follows:
The overall reaction for this process is:
Glucose 6-phosphate + 2 NADP+ + H2O → ribulose 5-phosphate + 2 NADPH + 2 H+ +
CO2
Non-oxidative phase
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
The pentose phosphate pathway's nonoxidative phase
Net reaction: 3 ribulose-5-phosphate → 1 ribose-5-phosphate + 2 xylulose-5-phosphate → 2
fructose-6-phosphate + glyceraldehyde-3-phosphate
Regulation
Glucose-6-phosphate dehydrogenase is the rate-controlling enzyme of this pathway. It is
allosterically stimulated by NADP+ and strongly inhibited by NADPH. The ratio of
NADPH:NADP+ is normally about 100:1 in liver cytosol. This makes the cytosol a highlyreducing environment. An NADPH-utilizing pathway forms NADP+, which stimulates Glucose6-phosphate dehydrogenase to produce more NADPH. This step is also inhibited by acetyl CoA.
G6PD activity is also post-translationally regulated by cytoplasmic deacetylase SIRT2. SIRT2mediated deacetylation and activation of G6PD stimulates oxidative branch of PPP to supply
cytosolic NADPH to counteract oxidative damage or support de novo lipogenesis.
Gluconeogenesis
Gluconeogenesis is a pathway consisting of a series of eleven enzyme-catalyzed reactions. The
pathway may begin in the mitochondria or cytoplasm (of the liver/kidney), this being dependent
on the substrate being used. Many of the reactions are the reverse of steps found in glycolysis.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr

Gluconeogenesis begins in the mitochondria with the formation of oxaloacetate by the
carboxylation of pyruvate. This reaction also requires one molecule of ATP, and is
catalyzed by pyruvate carboxylase. This enzyme is stimulated by high levels of acetylCoA (produced in β-oxidation in the liver) and inhibited by high levels of ADP and
glucose.

Oxaloacetate is reduced to malate using NADH, a step required for its transportation out
of the mitochondria.

Malate is oxidized to oxaloacetate using NAD+ in the cytosol, where the remaining steps
of gluconeogenesis take place.

Oxaloacetate is decarboxylated and then phosphorylated to form phosphoenolpyruvate
using the enzyme PEPCK. A molecule of GTP is hydrolyzed to GDP during this reaction.

The next steps in the reaction are the same as reversed glycolysis. However, fructose 1,6bisphosphatase converts fructose 1,6-bisphosphate to fructose 6-phosphate, using one
water molecule and releasing one phosphate (in glycolysis, phosphofructokinase 1
converts F6P and ATP to F1,6BP and ADP). This is also the rate-limiting step of
gluconeogenesis.

Glucose-6-phosphate is formed from fructose 6-phosphate by phosphoglucoisomerase
(the reverse of step 2 in glycolysis). Glucose-6-phosphate can be used in other metabolic
pathways or dephosphorylated to free glucose. Whereas free glucose can easily diffuse in
and out of the cell, the phosphorylated form (glucose-6-phosphate) is locked in the cell, a
mechanism by which intracellular glucose levels are controlled by cells.

The final reaction of gluconeogenesis, the formation of glucose, occurs in the lumen of
the endoplasmic reticulum, where glucose-6-phosphate is hydrolyzed by glucose-6phosphatase to produce glucose and release an inorganic phosphate. Like two steps prior,
this step is not a simple reversal of glycolysis, in which hexokinase catalyzes the
conversion of glucose and ATP into G6P and ADP. Glucose is shuttled into the
cytoplasm by glucose transporters located in the endoplasmic reticulum's membrane.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
Regulation
Glycolysis:
Glucose + 2 NAD+ + 2 ADP + 2 Pi  2 pyruvate + 2 NADH + 2 ATP
Gluconeogenesis:
2 pyruvate + 2 NADH + 4 ATP + 2 GTP  glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi
Glycolysis yields 2 ~P bonds of ATP.
Gluconeogenesis expends 6 ~P bonds of ATP and GTP.
A futile cycle consisting of both pathways would waste 4 ~P bonds per cycle.
To prevent this waste, Glycolysis and Gluconeogenesis pathways are reciprocally regulated.
Local Control includes reciprocal allosteric regulation by adenine nucleotides.

Phosphofructokinase (Glycolysis) is inhibited by ATP and stimulated by AMP.

Fructose-1,6-bisphosphatase (Gluconeogenesis) is inhibited by AMP.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
This insures that when cellular ATP is high (AMP would then be low), glucose is not degraded
to make ATP. When ATP is high it is more useful to the cell to store glucose as glycogen.
When ATP is low (AMP would then be high), the cell does not expend energy in synthesizing
glucose.
Global Control in liver cells includes reciprocal effects of a cyclic AMP cascade, triggered by
the hormone glucagon when blood glucose is low. Phosphorylation of enzymes and regulatory
proteins in liver by Protein Kinase A (cAMP-Dependent Protein Kinase) results in inhibition of
glycolysis and stimulation of gluconeogenesis, making glucose available for release to the blood.
Proteins relevant to these pathways that are phosphorylated by Protein Kinase A include:

Pyruvate Kinase, a glycolysis enzyme that is inhibited when phosphorylated.

CREB (cAMP response element binding protein) which activates, through other factors,
transcription
of
the
gene
for
PEP
Carboxykinase,
leading
to
increased
gluconeogenesis.

A bi-functional enzyme that makes and degrades an allosteric regulator, fructose-2,6bisphosphate.
Reciprocal regulation by fructose-2,6-bisphosphate:

Fructose-2,6-bisphosphate stimulates Glycolysis.
o
Fructose-2,6-bisphosphate allosterically activates the Glycolysis enzyme
Phosphofructokinase.
o
Fructose-2,6-bisphosphate also activates transcription of the gene for
Glucokinase, the liver variant of Hexokinase that phosphorylates glucose to
glucose-6-phosphate, the input to Glycolysis.

Fructose-2,6-bisphosphate
allosterically
inhibits
the
gluconeogenesis
Fructose-1,6-bisphosphatase.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
enzyme
Glycogen
Glycogen is a homopolymer made up of repeated units of α D glucose and each molecule is
linked to each other by 1→4 glycosidic bond which is a link connecting the 1st C atom of the
active glucose residue to the 6th C atom of the approaching glucose molecule. Once there is a
chain consisting of 8 to 10 glycosidic residues in the glycogen fragment, branching begins by
1→6 linkages. Liver glycogen is synthesized in well fed states. Muscle glycogen is synthesized
when the muscle glucose get depleted in intense physical exercise.
Chemical structure of glycogen
Glycogenesis Pathway
Glycogenenesis pathway is made up of series of steps resulting in the formation of complex
glycogen molecule from α D glucose in the cytoplasm of liver and muscle cells. .
Glycogenesis Steps
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
Gluconeogenesis Steps
UDP glucose – Synthesis of the carrier molecule:
UDP glucose acts as a vehicle that carries the glucose molecule which is to be added to the
budding glycogen molecule. UDP molecule and glucose 1 phosphate react in the presence of
UDP glucose pyrophosphorylase to form UDP glucose.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
Synthesis of UDP glucose
Glycogen primer
Glycogen synthesis cannot start from scratch. It needs a basic molecule on which the glucose
residues can be added so that the chain can get elongated. Glycogen fragments which already
exist can act as this primer. In glycogen depleted condition, a protein primer called glycogenin
acts as the flooring to which the glucose molecules from UDP glucose are added like bricks.
During the initial additions of glucose molecule, glycogenin acts as an auto catalyst and forms
the glycogen fragment on which further glucose residues are added by 1→4 linkage by the
enzyme glycogen synthase.
Elongation of glycogen chain:
The UDP glucose transfers the glucose molecule to the growing glycogen chain in such a way
that a link is formed between the 1st C atom of the standing glucose residue on the end point of
the fragment and 4th carbon of the glucose residue that is being added to the fragment.
This forms the 1→ 4 glycogenic link. The enzyme catalysing this step is glycogen synthase.
Branching in glycogen
If Glycogenesis stops with the above steps, it is expected to create a long linear molecule similar
to that of starch in plant. But this is not the case. After around 8 residues, branching begins and
the branches provide more number of activated glucose residual ends for the UDP glucose to get
attached to. This results in a highly branched easily soluble glycogen molecule. This branching is
brought about by branching enzyme called amylo-α(1→4) → α(1→6)-transglucosidase.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
The function of this enzyme is to break a fragment of glycosyl residues at the 1→ 4 linkage and
attach them to another glucose molecule on the chain, to form the branching points, by α(1→6)
linkage. This results in more number of end points for UDPglucose to add further glucose
residues to it. Thus branching enzyme results in extensively branched large glycogen molecule.
Defect in glycogen synthesis and glycogen degradation results in accumulation of abnormal
glycogen inside a cell which leads to glycogen storage disorders. One such genetic disease is
Glycogen storage disorder type 4 called as Anderson disease caused by defective branching
enzyme. So the glycogen formed is a linear insoluble structure that accumulates in the cells
causing liver and muscle damage.
Branched glycogen vs. linear starch
Regulation of Glycogenesis
Glycogen synthesis is strictly monitored to regulate the blood glucose level. It is activated in well
fed state and suppressed in fasting. According to basis of regulation of metabolic process, the
factors regulating Glycogenesis are
Availability of substrate
In well-fed state, when the blood glucose level is high, glucose 6 phosphate the substrate for
UDP glucose is also high. This allosterically increases Glycogenesis. Also during fasting, the
substrate is low and there is need for glucose which causes break down of glycogen which is
opposite of Glycogenesis.
Hormone:
Glycogen synthase, the key enzyme of Glycogenesis exists in activate (dephosphorylated) and
inactive (phosphorylated) form. Hormones like glucagon and epinephrine are diabetogenic i.e.
they increase the blood glucose level. Thus they antagonize glycogen synthesis which is an
effective way of reducing blood glucose level and storing it for further use.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
These hormones succeeds in their function by series of biochemical reactions which results in
phosphorylation of glycogen synthase enzyme rendering it inactive.
Insulin is an ant diabetic hormone. It lowers the blood glucose level by stimulating the uptake of
glucose by muscle cells and Glycogenesis in liver and muscle.
Glycogenolysis
Glycogenolysis is the breakdown of glycogen (n) to glucose-6-phosphate and glycogen (n-1).
Glycogen branches are catabolized by the sequential removal of glucose monomers via
phosphorolysis, by the enzyme glycogen phosphorylase.
Mechanism of Glycogen
The overall reaction for the breakdown of glycogen to glucose-1-phosphate is:
glycogen(n residues) + Pi ⇌ glycogen(n-1 residues) + glucose-1-phosphate
Here, glycogen phosphorylase cleaves the bond linking a terminal glucose residue to a glycogen
branch by substitution of a phosphoryl group for the α[1→4] linkage. Glucose-1-phosphate is
converted to glucose-6-phosphate by the enzyme phosphoglucomutase. Glucose residues are
phosphorolysed from branches of glycogen until four residues before a glucose that is branched
with a α[1→6] linkage. Glycogen debranching enzyme then transfers three of the remaining four
glucose units to the end of another glycogen branch. This exposes the α[1→6] branching point,
which is hydrolysed by α[1→6] glucosidase, removing the final glucose residue of the branch as
a molecule of glucose and eliminating the branch. This is the only case in which a glycogen
metabolite is not glucose-1-phosphate. The glucose is subsequently phosphorylated to glucose-6phosphate by hexokinase.
Function
Glycogenolysis takes place in the cells of the muscle and liver tissues in response to hormonal
and neural signals. In particular, glycogenolysis plays an important role in the fight-or-flight
response and the regulation of glucose levels in the blood.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
In myocytes (muscle cells), glycogen degradation serves to provide an immediate source of
glucose-6-phosphate for glycolysis, to provide energy for muscle contraction.
In hepatocytes (liver cells), the main purpose of the breakdown of glycogen is for the release of
glucose into the bloodstream for uptake by other cells. The phosphate group of glucose-6phosphate is removed by the enzyme glucose-6-phosphatase, which is not present in myocytes,
and the free glucose exits the cell via GLUT2 facilitated diffusion channels in the hepatocyte cell
membrane.
Regulation
Glycogenolysis is regulated hormonally in response to blood sugar levels by glucagon and
insulin, and stimulated by epinephrine during the fight-or-flight response. In myocytes, glycogen
degradation may also be stimulated by neural signals.
Disorders of Carbohydrate Metabolism
Carbohydrates are sugars. Some sugars are simple, and others are more complex. Sucrose (table
sugar) is made of two simpler sugars called glucose and fructose. Lactose (milk sugar) is made
of glucose and galactose. Both sucrose and lactose must be broken down into their component
sugars by enzymes before the body can absorb and use them. The carbohydrates in bread, pasta,
rice, and other carbohydrate-containing foods are long chains of simple sugar molecules. These
longer molecules must also be broken down by the body. If an enzyme needed to process a
certain sugar is missing, the sugar can accumulate in the body, causing problems.
Glycogen Storage Diseases
Glycogen storage diseases occur when there is a defect in the enzymes that are involved in the
metabolism of glycogen, resulting in growth abnormalities, weakness, and confusion.

Glycogen storage diseases are caused by lack of an enzyme needed to change glucose
into glycogen and break down glycogen into glucose.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr

Typical symptoms include weakness, sweating, confusion, kidney stones, and stunted
growth.

The diagnosis is made by examining a piece of tissue under a microscope (biopsy).

Treatment depends on the type of glycogen storage disease and usually involves
regulating the intake of carbohydrates.
Glycogen is made of many glucose molecules linked together. The sugar glucose is the body’s
main source of energy for the muscles (including the heart) and brain. Any glucose that is not
used immediately for energy is held in reserve in the liver, muscles, and kidneys in the form of
glycogen and is released when needed by the body.
There are many different glycogen storage diseases (also called glycogenoses), each identified
by a roman numeral. These diseases are caused by a hereditary lack of one of the enzymes that is
essential to the process of forming glucose into glycogen and breaking down glycogen into
glucose. About 1 in 20,000 infants has some form of glycogen storage disease.
Symptoms
Some of these diseases cause few symptoms. Others are fatal. The specific symptoms, age at
which symptoms start, and their severity vary considerably among these diseases. For types II,
V, and VII, the main symptom is usually weakness. For types I, III, and VI, symptoms are low
levels of sugar in the blood and protrusion of the abdomen (because excess or abnormal
glycogen may enlarge the liver). Low levels of sugar in the blood cause weakness, sweating,
confusion, and sometimes seizures and coma. Other consequences for children may include
stunted growth, frequent infections, and sores in the mouth and intestines.
Glycogen storage diseases tend to cause uric acid (a waste product) to accumulate in the joints,
which can cause gout (see Gout), and in the kidneys, which can cause kidney stones. In type I
glycogen storage disease, kidney failure is common in the second decade of life or later.
Diagnosis and Treatment
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
The specific type of glycogen storage disease is diagnosed by examining a piece of muscle or
liver tissue under a microscope (biopsy).
Treatment depends on the type of glycogen storage disease. For most types, eating many small
carbohydrate-rich meals every day helps prevent blood sugar levels from dropping. For people
who have glycogen storage diseases that cause low blood sugar levels, levels are maintained by
giving uncooked cornstarch every 4 to 6 hours around the clock. For others, it is sometimes
necessary to give carbohydrate solutions through a stomach tube all night to prevent low blood
sugar levels from occurring at night.
Types and Characteristics of Glycogen Storage Diseases
Affected Organs,
Name
Tissues, or Cells
Type O
von
Liver or muscle
Gierke’s
disease (type IA)
Symptoms
Episodes of low blood sugar levels (hypoglycemia) during
fasting if the liver is affected
Enlarged liver and kidney, slowed growth, very low blood
Liver and kidney sugar levels, and abnormally high levels of acid, fats, and
uric acid in blood
Same as in von Gierke’s disease but may be less severe
Liver and white
Type IB
blood cells
Low white blood cell count, recurring infections, and
inflammatory bowel disease
Pompe’s disease
(type II)
Forbes’
All organs
disease Liver,
(type III)
and heart
Andersen’s
Liver,
Enlarged liver and heart and muscle weakness
muscle, Enlarged liver or cirrhosis, low blood sugar levels, muscle
damage, heart damage, and weak bones in some people
muscle, Cirrhosis, muscle damage, and delayed growth and
disease (type IV) and most tissues
development
McArdle disease Muscle
Muscle cramps or weakness during physical activity
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Affected Organs,
Name
Tissues, or Cells
Symptoms
(type V)
Enlarged liver
Hers’
disease
(type VI)
Liver
Episodes of low blood sugar during fasting
Often no symptoms
Tarui’s
disease
(type VII)
Skeletal
and
cells
red
muscle
blood
Muscle cramps during physical activity and red blood cell
destruction (hemolysis)
Galactosemia
Galactosemia (a high blood level of galactose) is caused by lack of one of the enzymes necessary
for metabolizing galactose, a sugar present in lactose (milk sugar). A metabolite that is toxic to
the liver and kidneys builds up. The metabolite also damages the lens of the eye, causing
cataracts.

Galactosemia is caused by lack of one of the enzymes needed to metabolize the sugar in
milk.

Symptoms include vomiting, jaundice, diarrhea, and abnormal growth.

The diagnosis is based on a blood test.

Even with adequate treatment, affected children still develop mental and physical
problems.

Treatment involves completely eliminating milk and milk products from the diet.
Galactose is a sugar that is present in milk and in some fruits and vegetables. A deficient enzyme
or liver dysfunction can alter the metabolism, which can lead to high levels of galactose in the
blood (galactosemia). There are different forms of galactosemia, but the most common and the
most severe form is referred to as classic galactosemia.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
Symptoms
Newborns with galactosemia seem normal at first but, within a few days or weeks, lose their
appetite, vomit, become jaundiced, have diarrhea, and stop growing normally. White blood cell
function is affected, and serious infections can develop. If treatment is delayed, affected children
remain short and become intellectually disabled or may die.
Diagnosis
Galactosemia is detectable with a blood test. This test is done as a routine screening test for
newborns in all states in the United States. Before conception, adults with a sibling or child
known to have the disorder can be tested to find out whether they carry the gene that causes the
disease. If two carriers conceive a child, that child has a 1 in 4 chance of being born with the
disease.
Treatment
Galactosemia is treated by completely eliminating milk and milk products—the source of
galactose—from an affected child’s diet. Galactose is also present in some fruits, vegetables, and
sea products, such as seaweed. Doctors are not sure whether the small amounts in these foods
cause problems in the long term. People who have the disorder must restrict galactose intake
throughout life.
Mucopolysaccharidoses
Mucopolysaccharidoses are a group of hereditary disorders in which complex sugar molecules
are not broken down normally and accumulate in harmful amounts in the body tissues. The result
is a characteristic facial appearance and abnormalities of the bones, eyes, liver, and spleen,
sometimes accompanied by intellectual disability.

Mucopolysaccharidoses occur when the body lacks enzymes needed to break down and
store complex sugar molecules (mucopolysaccharides).

Typically, symptoms include short stature, hairiness, stiff finger joints, and coarseness of
the face.

The diagnosis is based on symptoms and a physical examination.

Although a normal life span is possible, some types cause premature death.

A bone marrow transplant may help.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
Complex sugar molecules called mucopolysaccharides are essential parts of many body tissues.
In mucopolysaccharidoses, the body lacks enzymes needed to break down and store
mucopolysaccharides. As a result, excess mucopolysaccharides enter the blood and are deposited
in abnormal locations throughout the body.
During infancy and childhood, short stature, hairiness, and abnormal development become
noticeable. The face may appear coarse. Some types of mucopolysaccharidoses cause intellectual
disability to develop over several years. In some types, vision or hearing may become impaired.
The arteries or heart valves can be affected. Finger joints are often stiff.
A doctor usually bases the diagnosis on the symptoms and a physical examination. The presence
of a mucopolysaccharidosis in other family members also suggests the diagnosis. Urine tests may
help but are sometimes inaccurate. X-rays may show characteristic bone abnormalities.
Mucopolysaccharidoses can be diagnosed before birth by using amniocentesis or chorionic villus
sampling
Prognosis and Treatment
The prognosis depends on the type of mucopolysaccharidosis. A normal life span is possible.
Some types, usually those that affect the heart, cause premature death.
In one type of mucopolysaccharidosis, attempts at replacing the abnormal enzyme have had
limited, temporary success. Bone marrow transplantation may help some people. However, death
or disability often results, and this treatment remains controversial.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
CHAPTER 2
Transamination
Introduction:
Transamination as the name implies, refers to the transfer of an amine group from one molecule
to another. This reaction is catalyzed by a family of enzymes called transaminases. Actually, the
transamination reaction results in the exchange of an amine group on one acid with a ketone
group on another acid. It is analogous to a double replacement reaction. The most usual and
major keto acid involved with transamination reactions is alpha-ketoglutaric acid, an
intermediate in the citric acid cycle. A specific example is the transamination of alanine to make
pyruvic acid and glutamic acid. Other amino acids which can be converted after several steps
through transamination into pyruvic acid include serine, cysteine, and glycine.
Transamination Reaction
Most amino acids are deaminated by transamination, a chemical reaction that transfers an
amino group to a ketoacid to form new amino acids. This is one of the major degradation
pathways which convert essential amino acids to nonessential amino acids (amino acids that can
be synthesized de novo by the organism).
Aminoacid + α-ketoglutarate ↔ α-keto acid + Glutamate
Glutamate's amino group, in turn, is transferred to oxaloacetate in a second transamination
reaction yielding aspartate.
Glutamate + oxaloacetate ↔ α-ketoglutarate + aspartate
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Mechanism of Action
Transamination catalyzed by aminotransferase occurs in two stages. In the first step, the α amino
group of an aminoacid is transferred to the enzyme, producing the corresponding α-keto acid and
the aminated enzyme. During the second stage, the amino group is transferred to the keto acid
acceptor, forming the amino acid product while regenerating the enzyme. The chirality of an
amino acid is determined during transamination. For the reaction to complete, aminotransferases
require participation of aldehyde containing coenzyme, pyridoxyl-5'-phosphate (PLP), a
derivative of Pyridoxine (Vitamin B6). The amino group is accommodated by conversion of this
coenzyme to pyridoxamine-5'-phosphate (PMP). PLP is covalently attached to the enzyme via
a Schiff Base linkage formed by the condesation of its aldehyde group with the ε-amino group of
an enzymatic Lys residue. The schiff base, which is conjugated to the enzymes pyridinium ring
is the focus of the coenzyme activity.
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Aminotransferase reaction occurs in two stages consisting of three steps: Transimination,
Tautomerisation and Hydolysis. In the first stage, alpha amino group of the aminoacid is
transferred to PLP yielding an alpha ketoacid and PMP. In the second stage of the
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reaction, in which the amino group of PMP is transferred to a different alpha Ketoacid to
yield a new alpha amino acid and PLP.
The product of transamination reactions depend on the availability of α-keto acids. The
products usually are either alanine, aspartate or glutamate, since their corresponding
alpha-keto acids are produced through metabolism of fuels. Being a major degradative
aminoacid pathway, lysine proline and threonine are the only three amino acids that do
not always undergo transamination and rather use respective dehydrogenase.
Alternative Mechanism
A second type of transamination reaction can be described as a nucleophilic substitution
of one amine or amide anion on an amine or ammonium salt. For example, the attack of a
primary amine by a primary amide anion can be used to prepare secondary amines:
RNH2 + R'NH− → RR'NH + NH2−
Symmetric secondary amines can be prepared using Raney nickel (2RNH2 → R2NH +
NH3). And finally, quaternary ammonium salts can be dealkylated using ethanolamine:
R4N+ + NH2CH2CH2OH → R3N + RN+H2CH2CH2OH
Aminonaphthalenes also undergo transaminations.[2]
Other Transamination Reactions:
Aspartic acid can be converted into oxaloacetic acid, another intermediate of the citric acid cycle.
Other amino acids such as glutamine, histidine, arginine, and proline are first converted into
glutamic acid.
Glutamine and asparagine are converted into glutamic acid and aspartic acid by a simple
hydrolysis of the amide group.
All of the amino acids can be converted through a variety of reactions and transamination into a
keto acid which is a part of or feeds into the citric acid cycle. The interrelationships of amino
acids with the citric acid cycle are illustrated in the graphic on the left.
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Oxidative Deamination Reaction
Introduction:
Deamination is also an oxidative reaction that occurs under aerobic conditions in all tissues but
especially the liver. During oxidative deamination, an amino acid is converted into the
corresponding keto acid by the removal of the amine functional group as ammonia and the amine
functional group is replaced by the ketone group. The ammonia eventually goes into the urea
cycle.
Oxidative deamination occurs primarily on glutamic acid because glutamic acid was the end
product of many transamination reactions.
The glutamate dehydrogenase is allosterically controlled by ATP and ADP. ATP acts as an
inhibitor whereas ADP is an activator.
Glutamate + NAD+ → α-ketoglutarate + NH4+ + NADH + H+
The resulting NH4+ enters the urea cycle and α-ketoglutarate may be used in the transamination
or Krebs cycle. The mentioned reaction is fully reversible – glutamate can be synthesized from
α-KG and NH4+ .
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We can conclude that most of the amino acid undergoes transamination in its degradation and
that the majority of amino nitrogen from amino acids is directly or indirectly concentrated in the
molecule of glutamate / glutamine. Amino nitrogen is subsequently released in glutaminase and
glutamate dehydrogenase reaction.
Urea (ornithine) cycle
Ammonia toxicity
Ammonia is a polar substance freely passing through physical barriers, as well as the bloodbrain barrier. When its concentration increases in the body, balance of many important
reactions is altered. Consider the following examples:
Glutamate + NAD+ → α-ketoglutarate + NH4+
Glutamate + NH4+ + ATP → glutamine + ADP + Pi
When an excess of ammonia, the glutamine concentration is gradually increasing. But glutamine
formation also consumes α-ketoglutarate of the Krebs cycle – speed of this pathway is gradually
decreasing and thus the production of energy in cells. The plasma ammonia concentration should
not exceed 35 µmol / L. In the human body, most of the toxic ammonia is converted to urea by
reactions of urea cycle.
Reactions in urea cycle
Urea, a non-toxic compound, is transported via the bloodstream to the kidneys where it is
excreted with the urine. Urea cycle is located in the matrix of mitochondria and cytosol of liver
cells. This pathway is an energy-consuming process in which the three substrates enter –
ammonia, carbon dioxide (bicarbonate) and aspartate (its amino group). Mitochondrial
carbamoyl phosphate synthetase I is the regulatory enzyme. Ornithine cycle communicates with
the Krebs cycle via oxaloacetate and fumarate.
Urea formation involves five reactions:
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1) Carbamoyl phosphate formation is catalyzed by mitochondrial carbamoyl phosphate
synthetase I:
NH4+ + HCO3- + ATP → carbamoyl phosphate + 2 ADP + Pi
2) Citrulline formation is catalyzed by ornithine transcarbamoylase:
Ornithine + carbamoyl phosphate → citrulline + Pi
Citrulline is passed into the cytosol.
3) Argininosuccinate formation is catalyzed by argininosuccinate synthetase:
Citrulline + Asp + ATP → argininosuccinate + AMP + PPi
4) Argininosuccinate break down is catalyzed by argininosuccinate lyase:
Argininosuccinate → arginine + fumarate
5) Hydrolysis of arginine is catalyzed by arginase:
Arginine + H2O → ornithine + urea
Ornithine returns into the mitochondrial matrix.
The urea cycle is closely linked to the Krebs cycle – from emerging fumarate becomes aspartate.
How does this relationship work? The fumarate is first hydrated to malate which is converted to
oxaloacetate by oxidation. Enzyme aspartate aminotransferase catalyzes transamination between
glutamate and oxaloacetate, resulting aspartate enters ornithine cycle. Glutamate is produced by
transamination of degraded amino acids which transmit their amino groups on the αketoglutarate molecule.
Regulation of ornithine cycle
Carbamoyl phosphate synthetase I, the main regulatory enzyme of ornithine cycle, is activated
by N-acetylglutamate. Enzyme N-acetylglutamate synthetase catalyzes the reaction between
AcCoA and glutamate which produces N-acetylglutamate. The amino acid arginine increases the
enzyme activity. Transcription of urea cycle enzymes is increased in high-protein diet or by
increasing protein catabolism (e.g. starvation), therefore in the increased supply of amino
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acids. The urea cycle belongs among proton-producing reactions, its activity is reduced at lower
pH – acidosis.
Degradation of amino acid carbon skeletons
Proteins in the human body contain 20 (21 if we include selenocysteine) proteinogenic amino
acids. Twenty (twenty-one) different multienzyme sequences exist for catabolism of amino acid
carbon skeletons. In this text we restrict ourselves only to the basic general mechanisms of amino
acid carbon skeleton degradation and a few selected examples.
Catabolism of amino acid carbon skeletons results in the formation of seven products: pyruvate,
acetyl-CoA, acetoacetyl-CoA, α-ketoglutarate, suc-CoA, fumarate and oxaloacetate. They have a
different fate in the energy metabolism. The strategy of the cell is to convert amino acid carbon
skeletons to compounds useful in gluconeogenesis or a molecule of lipids (fatty acids and ketone
bodies). Amino acids are divided into glucogenic and ketogenic amino acids according to the
fate of their degradation products. Amino acids leucine and lysine (starting with the letter L)
belong among ketogenic amino acids which lead to the formation of acetyl-CoA and acetoacetylCoA. Glucogenic amino acids include those that lead to the formation of the remaining five
products – pyruvate, α-ketoglutarate, suc-CoA, fumarate or oxaloacetate – serine, threonine,
cysteine, methionine, aspartate, glutamate, asparagine, glutamine, glycine, alanine, valine,
proline, histidine and arginine. But some amino acids have two degradation products – one of
them being glucogenic and second one ketogenic. These amino acids are called keto- and
glucogenic amino acids – they include isoleucine, phenylalanine, tyrosine and tryptophan.
The following overview shows degradation products of particular amino acids:
1) Acetyl-CoA and acetoacetyl-CoA – Lys and Leu are purely ketogenic amino acids, some
other amino acids (Phe, Tyr, Trp, Ile) provide glucogenic and ketogenic degradation products
2) α-ketoglutarate – five-carbon amino acids – Glu, Gln, Pro, Arg a His
3) Suc-CoA - nonpolar amino acids – Met, Ile a Val
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4) Fumarate - Phe, Tyr
5) Oxaloacetate – four-carbon amino acids – Asp a Asn
6) Pyruvate – Cys, Ala, Ser, Gly, Thr, Trp
Degradation of phenylalanine and tyrosine
Aromatic rings are quite stable, and therefore some brute force is needed to crack them open.
The best tool for this task is molecular oxygen, and a liberal dose of it is used in the breakdown
of phenylalanine. The pathway involves the following reactions:
1. Phenylalanine is converted to tyrosine by phenylalanine hydroxylase. In this reaction,
the second oxygen atom is released as water, reduced at the expense of the redox
cosubstrate tetrahydrobiopterin (BH4).
2. Tyrosine transaminase yields p-hydroxyphenylpyruvate.
3. p-Hydroxyphenylpyruvate dioxygenase uses another oxygen molecule to turn
hydroxyphenylpyruvate into homogentisate. I have traced the bits and pieces in this
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reaction using colors, but an actual explanation of the advanced magic performed by
the enzyme is beyond my feeble powers of narration.
4. Ring cleavage by homogentisate dioxygenase uses a third molecule of oxygen. The
product is maleylacetoacetate.
5. Maleylacetoacetate isomerase produces fumarylacetoacetate.
6. Fumarylacetoacetate hydrolase releases fumarate and acetoacetate.
Since the first transformation of phenylalanine yields tyrosine, it follows that the pathway
accounts for the degradation of both amino acids, and moreover that tyrosine is not an essential
amino acid. Of the two final products, fumarate can enter gluconeogenesis, while acetoacetate
cannot.
Degradation of Methionine
The degradation of methionine requires 9 steps. One of which involves the synthesis of
Sadenoylmethionine (SAM). The methyl group of SAM is highly reactive making it an important
methylating reagent. SAM is a common methyl-group donor in the cell. The first step is
catalyzed by methionine adenosyl transferase which tranfers the adenosyl group of ATP to the
sulfer of methionine to form SAM. Sam methylase transfers the activated methyl group to an
acceptor to form S-adenosylhomocysteine which is hydrolyzed by adenosylhomocysteinase to
form homocysteine. Cystathionine β-synthase is a PLP dependent enzyme that catalyzes the
condensation of a serine residue with homocysteine to form cystathionine.
Cystathioniine γ-lyase cleaves cystathionine into cysteine and α-ketobutyrate. α−ketobutyrate is
converted into propionyl CoA by α-ketobutyrate dehydrogenase which catalyzes a reaction that
is analogous to pyruvate dehydrogenase and α-ketoglutarate dehydrogenase.
Important derivatives of individual amino acids
Decarboxylation – formation of biogenic amines
Some amino acids undergo decarboxylation (removal of carboxyl group). The result is the
formation of biogenic amines (monoamines) that exhibit a broad spectrum of functions in the
human body. Here is the basic outline:
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1) Tyr → catecholamines (DOPA →dopamine → noradrenaline (norepinephrine) → adrenaline
(epinephrine)
2) Trp → serotonin (5-hydroxytryptamine)
3) Glu → γ-aminobutyrate (GABA)
4) His → histamine
5) Ser → ethanolamine → choline → acetylcholine
6) Cys → cysteamine
7) Asp → β-alanine
Nitric oxide (NO)
Nitric oxide is a vasodilator substance produced by endothelial cells. It is formed from Larginine in the reaction catalyzed by nitric oxide synthase (NO-synthase).
Hereditary enzyme defects in amino acid metabolism
Since there are so many different pathways for the degradation of the various amino acids, it is
understandable that many of the known inborn errors of metabolism are related to amino acid
metabolism. We will consider a few examples that affect the pathways discussed here.
Phenylketonuria (PKU)
 homozygous defect of phenylalanine hydroxylase
 affects one in 10,000 newborns among Caucasians; frequency differs with race
 excess of phenylalanine causes symptoms only after birth; intrauterine development
normal
 cognitive and neurological deficits, probably due to cerebral serotonin deficit
 treatment with phenylalanine-restricted diet
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 some cases are due to reduced affinity of enzyme for cofactor THB, can be treated
with high dosages of THB
As with most genetic enzyme defects, the clinical disease is manifest only in homozygous
individuals. Dietary phenylalanine that is not used for protein synthesis accumulates and causes
toxicity. It appears that the excess phenylalanine crowds out tryptophan at the L-aromatic amino
acid transporter in brain capillaries. This transporter keeps the brain supplied with all aromatic
amino acids. Since tryptophan is the precursor of the neurotransmitter serotonin, the competitive
inhibition of its transport to the brain results in a lack of cerebral serotonin which is believed to
cause the observed deficits in brain function and development.
In addition to phenylalanine itself, some aberrant metabolites derived from it also occur at
increased levels, and the appearance of ketone derivatives such as phenylpyruvic acid in the
urine has given the disease its name. These metabolites have no proven connection to the
pathogenesis of the disease.
Treatment of phenylketonuria
The treatment of phenylketonuria is limitation of dietary phenylalanine. Tyrosine is sufficiently
available in a reasonably protein-rich diet, so that the lack of its endogenous formation won’t be
a problem. The challenge, then, is to diagnose the disease in newborn kids, before any damage is
done. Happily, the enzyme defect does not cause a problem during fetal development, since the
placenta constantly equilibrates both useful and potentially harmful metabolites between the
maternal and the fetal circulation. Buildup of a metabolite in the fetus will therefore not occur as
long as the mother’s metabolism is able to degrade it.
Tyrosinemia
 homozygous defect of fumarylacetoacetate hydrolase
 fumarylacetoacetate and preceding metabolites back up
 fumaryl- and maleylacetoacetate react with glutathione and other nucleophiles,
causing liver toxicity
 the drug NTCB inhibits p-hydroxyphenylpyruvate dioxygenase, intercepting the
degradative pathway upstream of the toxic metabolites
 dietary restriction of tyrosine required to prevent neurological deficit
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Tyrosinemia is comparatively common in Quebec. In this case, there seems to be no
heterozygote advantage; instead, the high incidence is due to the so-called founder effect, that is,
the common descent of the afflicted population from a small group of founding settlers that
happened to contain one or several carriers of the gene.
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CHAPTER 3
Lipids
Lipids are naturally occurring organic compounds, commonly known as oils and fats. Lipids
occur through out the living world in microorganisms, higher plants and animals and also in all
cell types. Lipids contribute to cell structure, provide stored fuel and also take part in many
biological processes.
Lipids are naturally occurring hydrophobic molecules. They are heterogenous group of
compounds related to fatty acids. They include fats, oils, waxes, phospholipids, etc. They make
up about 70% of the dry weight of the nervous system. Lipids are crucial for the
healthy functioning of the nerve cells. Lipids are greasy or oily organic substances; lipids are
sparingly soluble in water and are soluble in organic solvents like chloroform, ether and benzene.
General characters of lipids

Lipids are relatively insoluble in water.

They are soluble in non-polar solvents, like ether, chloroform, methanol.

Lipids have high energy content and are metabolized to release calories.

Lipids also act as electrical insulators, they insulate nerve axons.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr

Fats contain saturated fatty acids, they are solid at room temperatures. Example, animal
fats.

Plant fats are unsaturated and are liquid at room temperatures.

Pure fats are colorless, they have extremely bland taste.

The fats are sparingly soluble in water and hence are described are hydrophobic
substances.

They are freely soluble in organic solvents like ether, acetone and benzene.

The melting point of fats depends on the length of the chain of the constituent fatty acid
and the degree of unsaturation.

Geometric isomerism, the presence of double bond in the unsaturated fatty acid of the
lipid molecule produces geometric or cis-trans isomerism.

Fats have insulating capacity, they are bad conductors of heat.

Emulsification is the process by which a lipid mass is converted to a number of small
lipid droplets. The process of emulsification happens before the fats can be absorbed by
the intestinal walls.

The fats are hydrolyzed by the enzyme lipases to yield fatty acids and glycerol.

The hydrolysis of fats by alkali is called saponification. This reaction results in the
formation of glycerol and salts of fatty acids called soaps.

Hydrolytic rancidity is caused by the growth of microorganisms which secrete enzymes
like lipases. These split fats into glycerol and free fatty acids.
Classification of Lipids
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Simple Lipids or Homolipids
Simple lipids are the esters of fatty acids with various alcohols.
Fats and Oils (triglycerides and triacylglycerols) - These are esters of fatty acids with a
trihydroxy alcohol, glycerol. A fat is solid at ordinary room temperature, an oil is liquid.
Simple Triglycerides - Simple triglycerides are one in which three fatty acids radicles are similar
or are of the same type. Example: Tristearin, Triolein.
Mixed Triglycerides are one in which the three fatty acids radicles are different from each other.
Example: distearo-olein, dioleo-palmitin.
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Waxes are the esters of fatty acids with high molecular weight monohydroxy alcohols. Example:
Beeswax, Carnauba wax.
Compound Lipids or Heterolipids
Heterolipids are esters of fatty acids with alcohol and possess additional groups also.
Phospholipids or Phosphatids are compound containing fatty acids and glycerol in addition to a
phosphoric acid, nitrogen bases and other substituents. They usually possess one hydrophilic
head and tow non-polar tails. They are called polar lipids and are amphipathic in nautre.
Phospholipids can be phosphoglycerides, phosphoinositides and phosphosphingosides.
Phosphoglycerides are major phospholipids, they are found in membranes. It contains fatty acid
molecules which are esterified to hydroxyl groups of glycerol. The glycerol group also forms an
ester
linkage
with
phosphoric
acid.
Example:
Lecithin,
Cephalins.
Phosphoinositides are said to occur in phospholipids of brain tissue and soybeans. The ply
important
role
in
transport
processes
in
cells.
Phosphosphingosides are commonly found in nerve tissue. Example: sphingomyelins.
Glycolipids are the compounds of fatty acids with carbohydrates and contain nitrogen but no
phosphoric acid. The glycolipids also include certain structurally related compounds comprising
the groups gangliosides, sulpholipids and sulfatids.
Derived Lipids
Derived lipids are the substances derived from simple and compound lipids by hydrolysis. These
includes fatty acids, alcohols, monoglycerides and diglycerides, steroids, terpenes, carotenoids.
The most common derived lipids are steroids, terpenes and carotenoids.
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Steroids do not contain fatty acids, they are nonsaponifiable, and are not hydrolyzed on heating.
They are widely distributed in animals, where they are associated with physiological processes.
Example: Estranes, androstranes, etc.
Terpenes in majority are found in plants. Example: Natural rubber. gernoil, etc.
Carotenoids are tetraterpenes. They are widely distributed in both plants and animals. They are
exclusively of plant origin. Due to the presence of many conjugated double bonds, they are
colored red or yellow. Example: Lycopreene, carotenes, Xanthophylls.
Essential fatty acids are those that cannot be constructed through any chemical pathways, known
to happen in humans. They must be obtained from the diet. Linoleic acid and linolenic acid are
the essential fatty acids.
Non-essential fatty acids are those which are not necessary to be taken through diet, they are
synthesized through chemical pathways.
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Unsaturated fatty acids have one or more double bonds between carbon atoms. The tow carbon
atoms are bound to each other through double bonds and can occur in cis or trans configuration.
Saturated fatty acids are long chain carboxylic acids and do not have double bonds. Example:
Arachidic acid, Palmitic acid, etc.
Structure of Lipids
Lipids has no single common structure. The most commonly occurring lipids are triglycerides
and phospholipids.
Triglycerides are fats and oils. Triglycerides have a glycerol backbone bonded to three fatty
acids. If the three fatty are similar then the triglyceride is known as simple triglyceride. If the
fatty acids are not similar then the fatty acids are known as mixed triglyceride.
The second most common class of lipids are phospholipids. They are found in membranes
of animal and plants. Phospholipids contains glycerol and fatty acids, they also contain
phosphoric acids and a low-molecular weight alcohol. Common phospholipids are lecithins and
cephalins.
Function of Lipids
Lipids perform several biological functions:

Lipids are storage compounds, triglycerides serve as reserve energy of the body.

Lipids are important component of cell membranes structure in eukaryotic cells.

Lipids regulate membrane permeability.

They serve as source for fat soluble vitamins like A, D, E, K.

They act electrical insulators to the nerve fibres, where the myelin sheath contains
lipids.

Lipids are components of some enzyme systems.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr

Some lipids like prostaglandins and steroid hormones act as cellular metabolic
regulators.

Cholesterol is found in cell membranes, blood, and bile of many organisms.

As lipids are small molecules and are insoluble in water, they act as signalling
molecules.

Layers of fat in the subcutaneous layer, provides insulation and protection from cold.
Body temperature maintenance is done by brown fat.

Polyunsaturated phospholipids are important constituents of phospholipids, they provide
fluidity and flexibility to the cell membranes.

Lipoproteins that are complexes of lipids and proteins, occur in blood as plasma
lipoprotein, they enable transport of lipids in aqueous environment, and their transport
throughout the body.

Cholesterol maintains fluidity of membranes by interacting with lipid complexes.

Cholesterol is the precursor of bile acids, Vitamin D and steroids.

Essential fatty acids like linoleic and linolenic acids are precursors of many different
types of ecosanoids including prostaglandins, thromboxanes. These play a important role
in pain, fever, inflammation and blood clotting.
Cholesterol
Cholesterol is a well-studied lipid, because of its strong correlation with the incidence
cardiovascular disease. It is an important component of cell membranes and plasma
lipoproteins, and is an important precursor of many biologically important substances like
bile acids and steroid hormones. It is abundant in nerve tissues and is associated with
gallstones.
Dietary cholesterol is found in saturated fats of animals (as butter and lard), but vegetable
oils do not contain cholesterol. Only a small portion of your body cholesterol comes from
the diet. Most of it is produced in the body. Eating unsaturated fatty acids from vegetable
oil helps lower blood cholesterol levels by reducing cholesterol synthesis in the body.
However, eating saturated fats from animal fat elevates blood cholesterol and
triglycerides and reduce the ratio of your good to bad cholesterol.
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TERPENS
Introduction
Terpenoids (or isoprenoids), a subclass of the prenyllipids (terpenes, prenylquinones, and
sterols), represent the oldest group of small molecular products synthesized by plants and are
probably
the
most
widespread
group
of
natural
products.
Terpenoids can be described as modified terpenes, where methyl groups are moved or removed,
or oxygen atoms added. Inversely, some authors use the term "terpenes" more broadly, to include
the terpenoids.
Structure and biosynthesis
Isoprene
Terpenes are derived biosynthetically from units of isoprene, which has the molecular formula
C5H8. The basic molecular formulae of terpenes are multiples of that, (C5H8)n where n is the
number of linked isoprene units. This is called the isoprene rule or the C5 rule. The isoprene
units may be linked together "head to tail" to form linear chains or they may be arranged to form
rings. One can consider the isoprene unit as one of nature's common building blocks.
Classifications of Terpenes
Terpenes may be classified by the number of isoprene units in the molecule; a prefix in the name
indicates the number of terpene units needed to assemble the molecule.

Hemiterpenes consist of a single isoprene unit. Isoprene itself is considered the only
hemiterpene, but oxygen-containing derivatives such as prenol and isovaleric acid are
hemiterpenoids.

Monoterpenes consist of two isoprene units and have the molecular formula C10H16.
Examples of monoterpenes and monoterpenoids include geraniol, terpineol (present in
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lilacs), limonene (present in citrus fruits), myrcene (present in hops), linalool (present in
lavender) or pinene (present in pine trees).[7] Iridoids derive from monoterpenes.

Sesquiterpenes consist of three isoprene units and have the molecular formula C15H24.
Examples of sesquiterpenes and sesquiterpenoids include humulene, farnesenes, farnesol.
(The sesqui- prefix means one and a half.)

Diterpenes are composed of four isoprene units and have the molecular formula C20H32.
They derive from geranylgeranyl pyrophosphate. Examples of diterpenes and
diterpenoids are cafestol, kahweol, cembrene and taxadiene (precursor of taxol).
Diterpenes also form the basis for biologically important compounds such as retinol,
retinal, and phytol.

Sesterterpenes, terpenes having 25 carbons and five isoprene units, are rare relative to
the other sizes. (The sester- prefix means half to three, i.e. two and a half.) An example of
a sesterterpenoid is geranylfarnesol.

Triterpenes consist of six isoprene units and have the molecular formula C30H48. The
linear triterpene squalene, the major constituent of shark liver oil, is derived from the
reductive coupling of two molecules of farnesyl pyrophosphate. Squalene is then
processed biosynthetically to generate either lanosterol or cycloartenol, the structural
precursors to all the steroids.

Sesquarterpenes are composed of seven isoprene units and have the molecular formula
C35H56. Sesquarterpenes are typically microbial in their origin. Examples of
sesquarterpenoids are ferrugicadiol and tetraprenylcurcumene.

Tetraterpenes contain eight isoprene units and have the molecular formula C40H64.
Biologically important tetraterpenoids include the acyclic lycopene, the monocyclic
gamma-carotene, and the bicyclic alpha- and beta-carotenes.

Polyterpenes consist of long chains of many isoprene units. Natural rubber consists of
polyisoprene in which the double bonds are cis. Some plants produce a polyisoprene with
trans double bonds, known as gutta-percha.

Norisoprenoids, such as the C13-norisoprenoids 3-oxo-α-ionol present in Muscat of
Alexandria leaves and 7,8-dihydroionone derivatives, such as megastigmane-3,9-diol and
3-oxo-7,8-dihydro-α-ionol found in Shiraz leaves (both grapes in the species Vitis
vinifera) or wine can be produced by fungal peroxidases or glycosidases.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
They are universally present in small amounts in living organisms, where they play numerous
vital roles in plant physiology as well as important functions in all cellular membranes. On the
other hand, they are also accumulated in many cases, and it is shown that the extraordinary
variety they then display can be due to ecological factors playing an evolutionary role. Monoand sesquiterpenes are the chief constituents of the essential oils while the other terpenes are
constituents of balsams, resins, waxes, and rubber.
Oleoresin is a roughly equal mixture of turpentine (85% C10-monoterpenes and 15% C15sesquiterpenes) and rosin (C20-diterpene) that acts in many conifer species to seal wounds and is
toxic to both invading insects and their pathogenic fungi. A number of inducible terpenoid
defensive compounds (phytoalexins) from angiosperm species are well known. These include
both sesquiterpenoid and diterpenoid types.
Isoprenoid units are also found within the framework of other natural molecules. Thus, indole
alkaloids, several quinones (vitamin K), alcohols (vitamin E, vitamin A formed from
-
carotene), phenols, isoprenoid alcohols (also known as terpenols or polyprenols) also contain
terpenoid fragments.
Prostaglandins
Introduction:
Prostaglandins were first discovered and isolated from human semen in the 1930s by Ulf von
Euler of Sweden. Thinking they had come from the prostate gland, he named them
prostaglandins. It has since been determined that they exist and are synthesized in virtually every
cell of the body.
Prostaglandins, are like hormones in that they act as chemical messengers, but do not move to
other sites, but work right within the cells where they are synthesized. Prostaglandins are
unsaturated carboxylic acids, consisting of of a 20 carbon skeleton that also contains a five
member ring. They are biochemically synthesized from the fatty acid, arachidonic acid. See the
graphic on the left. The unique shape of the arachidonic acid caused by a series of cis double
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
bonds helps to put it into position to make the five member ring. See the prostaglandin in the
next panel.
Prostaglandin Structure:
Prostaglandins are unsaturated carboxylic acids, consisting of of a 20 carbon skeleton that also
contains a five member ring and are based upon the fatty acid, arachidonic acid. There are a
variety of structures one, two, or three double bonds. On the five member ring there may also be
double bonds, a ketone, or alcohol groups. A typical structure is on the left graphic.
Functions of Prostaglandins:
There are a variety of physiological effects including:
1. Activation of the inflammatory response, production of pain, and fever. When tissues are
damaged, white blood cells flood to the site to try to minimize tissue destruction. Prostaglandins
are produced as a result.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
2. Blood clots form when a blood vessel is damaged. A type of prostaglandin called
thromboxane stimulates constriction and clotting of platelets. Conversely, PGI2, is produced to
have the opposite effect on the walls of blood vessels where clots should not be forming.
3. Certain prostaglandins are involved with the induction of labor and other reproductive
processes. PGE2 causes uterine contractions and has been used to induce labor.
4. Prostaglandins are involved in several other organs such as the gastrointestinal tract (inhibit
acid synthesis and increase secretion of protective mucus), increase blood flow in kidneys, and
leukotriens promote constriction of bronchi associated with asthma.
Effects of Aspirin and other Pain Killers:
When you see that prostaglandins induce inflammation, pain, and fever, what comes to mind but
aspirin. Aspirin blocks an enzyme called cyclooxygenase, COX-1 and COX-2, which is involved
with the ring closure and addition of oxygen to arachidonic acid converting to prostaglandins.
The acetyl group on aspirin is hydrolzed and then bonded to the alcohol group of serine as an
ester. This has the effect of blocking the channel in the enzyme and arachidonic can not enter the
active site of the enzyme.
By inhibiting or blocking this enzyme, the synthesis of prostaglandins is blocked, which in turn
relives some of the effects of pain and fever.
Aspirin is also thought to inhibit the prostaglandin synthesis involved with unwanted blood
clotting in coronary heart disease. At the same time an injury while taking aspirin may cause
more extensive bleeding.
See the following chime tutorial for the detailed molecular basis for the inhibition of the COX
enzyme by aspirin.
OXIDATION OF LONG-CHAIN FATTY ACIDS
Two Forms of Carrying Fatty Acids:

PLASMA ALBUMIN: Can carry up to 10 molecules of fats in the blood serum.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
o
Also carries varies drugs and pharmacological agents. The albumin capacity for
carrying these drugs must be considered with polypharmacy.
ACTIVATION OF FATTY ACIDS: Fats are delivered to cells as free fats. They must be
activated before they can be burned.

Acyl-CoA Synthetase: Free Fat ------> Acyl-CoA Thioester, which has a high-energy
bond.
o ATP is required in the synthesis.
o This step is fully reversible, as ATP and the Acyl-CoA Thioester product both
have equivalent energy levels.
o To prevent the reversibility, the reaction is coupled to Pyrophosphatase, which
catalyzes Pyrophosphate ------> 2 Inorganic Phosphate, which breaks a high
energy bond to drive the reaction to the right.
TRANSLOCATION OF FATTY ACYL-CoA THIOESTER: The Acyl-CoA must get into the
mitochondrial matrix.


Once activated, the Acyl-CoA can get through the out mitochondrial membrane by
traversing through a Porin protein.
Carnitine Intermediate: Only Long-chain fatty acids are converted to carnitine as an
intermediate. Short-chain fats can traverse the inner membrane directly:
o INTERMEMBRANE SPACE: Carnitine Acyl Transferase I: Acyl-CoA ------>
Acyl Carnitine.
 Carnitine is a simpler structure than Coenzyme-A. The fat is esterified to
carnitine, temporarily, for the purpose of transport.
o Translocase: Only recognizes Acyl-Carnitine. It translocated the carnitine
structure through the inner membrane to the matrix.
o MITOCHONDRIAL MATRIX: Carnitine Acyl Transferase II: Acyl-Carnitine -----> Acyl-CoA
o In the matrix the fat is esterified back to Coenzyme-A.
beta-OXIDATION: A four-step process. Called beta-Oxidation because most of the chemistry
occurs on the beta-Carbon (beta to the carbonyl) per turn of the cycle.

The Four Steps: Ultimately we are oxidizing the beta-Carbon from most reduced to most
oxidized state.
o OXIDATION: Acyl-CoA Dehydrogenase catalyzes an elimination of hydrogens
on the alpha-carbon, to create the alpha,beta-Unsaturated Acyl-CoA.
 FAD ------> FADH2 is the corresponding reduction.
o HYDRATION: Add water across the double bond, creating an OH group on the
beta-Carbon.
o OXIDATION: Oxidize the OH group to a carbonyl function. Now we have a
beta-keto-acid
+
 NAD ------> NADH is the corresponding reduction.
o THIOLYSIS: Cut the end-acid off, and add CoA to the newly created keto-group.
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An additional mole of Coenzyme-A is esterified to the beta-Keto
function, leaving Acetyl-CoA and an Acyl-CoA of two less carbons
The Ultimate Products: Every cycle of beta-Oxidation (1) reduces the fat-chain by two
carbons and (2) yields a free Acetyl-CoA (which can then be further metabolized as
directed).
o Acyl-CoA(n-2)
o Acetyl-CoA
Similarity to TCA Cycle: The final four steps of the TCA Cycle are nearly identical to
these in their chemistry.
o Succinate ------> Fumarate -- OXIDATION
o Fumarate ------> Malate -- HYDRATION
o Malate ------> Oxaloacetate -- OXIDATION
Odd Chain Fats: Most fats are even-numbered. But beta-Oxidation can occur with odd
chains, at which point the products are Acetyl-CoA (2C) and Propanoyl-CoA (3C).
Propanoyl-CoA is then metabolized by a different mechanism.




ENERGETICS OF beta-OXIDATION:



COST: -2 ATP, but we only have to invest that once!
o -1 ATP for the Acyl-CoA Ligase
o -1 ATP net for the Pyrophosphatase, since we actually end up with AMP.
BENEFIT: Per turn of beta-Oxidation (i.e. per two carbons).
o Acetyl-CoA +12 ATP
o FADH2 +2 ATP
o NADH +3 ATP
o Total +17 ATP per 2 carbon, or about 8 per carbon
By Comparison, Glucose gives us about +36 ATP per 6 carbons, or about 6 per carbon
REGULATION OF beta-OXIDATION:



Positive Effectors: Starvation and a general low-energy level
o Low insulin and high glucagon (i.e. low insulin:glucagon ratio)
o ADP
Malonyl-CoA inhibits it because it is a reactant of fat-synthesis.
o It inhibits the Carnitine Acetyltransferase, preventing the transport of fats into
the mitochondria and thereby effectively slowing beta-oxidation.
o This occurs in conjunction with fat-synthesis, so that newly synthesized fats are
not immediately broken down again.
Negative Effectors: General indicators of sufficient energy in the cell:
o High insulin and low glucagon
o High ATP
REFSUM'S DISEASE:

Phytanate = a 15-Carbon animal-fat, originally derived from Phytol in plants (a plant
alcohols essential to photosynthesis).
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
Phytanate cannot be metabolized because there is a methyl group on the betaCarbon, thus disturbing beta-oxidation (ya can't get the alpha,beta-unsaturated
intermediate).
o Normally this problem is fixed by breaking it down to a 14 carbon fat, Pristanate,
thus eliminating the beta-methyl group (i.e. it is now an alpha-methyl group).
Biochemical Basis of Refsum's Disease: We can't get from phytanate to pristanate.
o Consequence = buildup of phytanate especially in nervous tissues.
Symptoms:
o Retinitis Pigmentosum, Cerebellar ataxia, peripheral neuropathies, skeletal
abnormalities.
o


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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
Fatty Acid Biosynthesis

Synthesis takes place in the cytosol

Intermediates covalently linked to acyl carrier protein

Activation of each acetyl CoA.

acetyl CoA + CO2

Four-step repeating cycle, extension by 2-carbons /cycle
Malonyl CoA
– Condensation
– Reduction
– Dehydration
– reduction
Fatty acid synthesis
• The enzymes of fatty acid synthesis are packaged together in a complex called as fatty acid
synthase (FAS).
• The product of FAS action is palmitic acid. (16:0).
• Modifications of this primary FA leads to other longer (and shorter) FA and unsaturated FA.
• The fatty acid molecule is synthesized 2 carbons at a time
• FA synthesis begins from the methyl end and proceeds toward the carboxylic acid end. Thus,
C16 and C15 are added first and C2 and C1 are added last.
• C15 and C16 are derived directly from acetylCoA. For further step-wise 2-carbon extensions,
acetylCoA is first activated to malonyl CoA, a 3-carbon compound, by the addition of a CO2.
Citrate Shuttle
• FAs are synthesized in the cytoplasm from acetylCoA.
• AcetylCoA generated from pyruvate by the action of PDH and by β-oxidation of fatty acids is
in the mitochondria.
• For fatty acid biosynthesis, acetylCoA has to be transported from the mitochondria to the
cytoplasm. This is done via a shuttle system called the Citrate Shuttle.
• AcetylCoA reacts with oxaloacetate to give citrate. A tricarboxylate translocase transports
citrate from mitochondria to cytosol.
• In the cytosol, citrate is cleaved back to oxaloacetate and acetylCoA. This reaction is catalyzed
by ATP-citrate lyase and requires the hydrolysis of one molecule of ATP.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
Citrate Shuttle (regeneration of pyruvate)
• Oxaloacetate is converted back to pyruvate for re-entry into mitochondria
• Step 1. Oxaloacetate + NADH + H+
malate + NAD+. Reverse of the TA cycle reaction.
Catalyzed by cytosolic malate dehydrogenase.
• Step 2. Malate + NADP+
pyruvate + CO2 + NADPH. Catalyzed by malic enzyme
• Pyruvate translocase transports pyruvate into mitochondria.
• Pyruvate is converted to oxaloacetate by pyruvate carboxylase with coupled hydrolysis of one
ATP. Pyruvate + ATP + CO2 + H2O
oxaloacetate + ADP + Pi (reaction of gluconeogenesis)
• Net Reaction: NADP+ + NADH + H+ + ATP + H2O
NADPH +
NAD+ + ADP + Pi
• Thus, transport of acetylCoA to cytosol requires expense of one ATP and conversion of one
NADH to NADPH.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
MalonylCoA
• Malonyl CoA is synthesized by the action of acetylCoA carboxylase. Biotin is a required
cofactor.
• CH3COSCoA + CO2 + ATP
OOC-CH2-COSCoA+ ADP +Pi (enzyme: acetylCoA
carboxylase)
• This is an irreversible reaction. AcetylCoAcarboxylation is a rate-limiting step of FA
biosynthesis.
• AcetylCoA carboxylase is under allostericregulation. Citrate is a positive effector andpalmitoyl
CoA is a negative effector.
Fatty Acid Biosynthesis
Fatty Acid Synthase (FAS)
• FAS is a polypeptide chain with multiple domains, each with distinct enzyme activities
required for fatty acid biosynthesis.
• ACP: For fatty acid biosynthesis, the activator is a protein called the acyl carrier protein (ACP).
It is part of the FAS complex. The acyl groups get anchored to the CoA group of ACP by a
thioester linkage
• Condensing enzyme/β-ketoacyl synthase (K-SH). Also part of FAS, CE has a cysteine SH that
participates in thioester linkage with the carboxylate group of the fatty acid.
• During FA biosynthesis, the growing FA chain alternates between K-SH and ACP-SH
Step-wise reactions
1. The acetyl group gets transferred from CoA to ACP by acetyl CoA-ACP transacylase.
2. The acetyl (acyl) group next gets transferred to the K arm of FAS complex.
3. Next, the malonyl group gets transferred from CoA to ACP by malonyl CoA ACP
transacylase. This results in both arms of FAS occupied forming acylmalonyl- ACP.
4. The COO group of malonyl ACP is removed as CO2, the acetyl group (C16 and C15) gets
transferred to the alpha carbon of malonyl ACP. This results in 3- keto acyl ACP.
Reactions of FA biosynthesis
• The 3-keto group is converted to a CH2 by a series of reactions reverse to FA β-oxidation.
Namely,
1. reduction to hydroxyl group. Enz: 3-keto acyl ACP reductase
2. dehydration to form a 2,3 double bond and Enz: 3- hydroxy acyl ACP dehydratase
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
3. a second reduction to remove the double bond. Enz: Enoyl ACP reductase
• Both reduction reactions require the reduced cofactor NADPH.
• The result of the first cycle of fatty acid biosynthesis is a four carbon chain associated to the
ACP arm.
• This chain gets transferred to the K arm.
• A new malonyl CoA is introduced on the ACP arm.
• The reactions proceed as before. For each cycle the acyl group transferred to the α carbon of
malonyl CoA is 2- carbons longer the previous cycle.
• At the end of 7 cycles a 16 carbon chain is attached to the
ACP arm (palmitoyl ACP).
• The C16 unit is hydrolyzed from ACP yielding free palmitate
• Net reaction: Acetyl CoA + 7 malonyl CoA + 14 NADPH + 14 H+
Palmitate + 7 CO2 + 8
CoA + 14 NADP+ + 6H2O
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
Regulation of fatty acid synthesis
Acetyl-CoA is formed into malonyl-CoA by acetyl-CoA carboxylase, at which point malonylCoA is destined to feed into the fatty acid synthesis pathway. Acetyl-CoA carboxylase is the
point of regulation in saturated straight-chain fatty acid synthesis, and is subject to both
phosphorylation and allosteric regulation. Regulation by phosphorylation occurs mostly in
mammals, while allosteric regulation occurs in most organisms. Allosteric control occurs as
feedback inhibition by palmitoyl-CoA and activation by citrate. When there are high levels of
palmitoyl-CoA, the final product of saturated fatty acid synthesis, it allosterically inactivates
acetyl-CoA carboxylase to prevent a build-up of fatty acids in cells. Citrate acts to activate
acetyl-CoA carboxylase under high levels, because high levels indicate that there is enough
acetyl-CoA to feed into the Krebs cycle and produce energy.
High plasma levels of insulin in the blood plasma (e.g. after meals) cause the dephosphorylation
of acetyl-CoA carboxylase, thus promoting the formation of malonyl-CoA from acetyl-CoA, and
consequently the conversion of carbohydrates into fatty acids, while epinephrine and glucagon
(released into the blood during starvation and exercise) cause the phosphorylation of this
enzyme, inhibiting lipogenesis in favor of fatty acid oxidation via beta-oxidation.
Fatty acid catabolism
A diagrammatic illustration of the process of lipolysis (in a fat cell) induced by high epinephrine
and low insulin levels in the blood. Epinephrine binds to a beta-adrenergic receptor in the cell
wall of the adipocyte, which causes cAMP to be generated inside the cell. The cAMP activates a
protein kinase, which phosphorylates and thus, in turn, activates a hormone-sensitive lipase in
the fat cell. This lipase cleaves free fatty acids from their attachment to glycerol in the fat stored
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
in the fat droplet of the adipocyte. The free fatty acids and glycerol are then released into the
blood. However more recent studies have shown that adipose triglyceride lipase has to first
convert triacylglycerides to diacylglycerides, and that hormone-sensitive lipase converts the
diacylglycerides to monoglycerides and free fatty acids. Monoglycerides are hydrolyzed by
monoglyceride lipase.[3] The activity of hormone sensitive lipase is regulated by the circulation
hormones insulin, glucagon, norepinephrine, and epinephrine, as shown in the diagram.
A diagrammatic illustration of the transport of free fatty acids in the blood attached to plasma
albumin, its diffusion across the cell membrane using a protein transporter, and its activation,
using ATP, to form acyl-CoA in the cytosol. The illustration is, for diagrammatic purposes, of a
12 carbon fatty acid. Most fatty acids in human plasma are 16 or 18 carbon atoms long.
A diagrammatic illustration of the transfer of an acyl-CoA molecule across the inner membrane
of the mitochondrion by carnitine-acyl-CoA transferase (CAT). The illustrated acyl chain is, for
diagrammatic purposes, only 12 carbon atoms long. Most fatty acids in human plasma are 16 or
18 carbon atoms long. CAT is inhibited by high concentrations of malonyl-CoA (the first
committed step in fatty acid synthesis) in the cytoplasm. This means that fatty acid synthesis and
fatty acid catabolism cannot occur simultaneously in any given cell.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
A diagrammatic illustration of the process of the beta-oxidation of an acyl-CoA molecule in the
mitochodrial matrix. During this process an acyl-CoA molecule which is 2 carbons shorter than
it was at the beginning of the process is formed. Acetyl-CoA, water and 5 ATP molecules are the
other products of each beta-oxidative event, until the entire acyl-CoA molecule has been reduced
to a set of acetyl-CoA molecules.
Fatty acids are released, between meals, from the fat depots in adipose tissue, where they are
stored as triglycerides, as follows:

Lipolysis, the removal of the fatty acid chains from the glycerol to which they are bound
in their storage form as triglycerides (or fats), is carried out by lipases. These lipases are
activated by high epinephrine and glucagon levels in the blood (or norepinephrine
secreted by sympathetic nerves in adipose tissue), caused by declining blood glucose
levels after meals, which simultaneously lowers the insulin level in the blood.

Once freed from glycerol, the free fatty acids enter the blood, which transports them,
attached to plasma albumin, throughout the body.

Long chain free fatty acids enter the metabolizing cells (i.e. most living cells in the body
except red blood cells and neurons in the central nervous system) through specific
transport proteins, such as the SLC27 family fatty acid transport protein. Red blood cells
do not contain mitochondria and are therefore incapable of metabolizing fatty acids; the
tissues of the central nervous system cannot use fatty acids, despite containing
mitochondria, because fatty acids cannot cross the blood brain barrier into the interstitial
fluids that bathe these cells.

Once inside the cell long-chain-fatty-acid—CoA ligase catalyzes the reaction between a
fatty acid molecule with ATP (which is broken down to AMP and inorganic
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
pyrophosphate) to give a fatty acyl-adenylate, which then reacts with free coenzyme A to
give a fatty acyl-CoA molecule.

In order for the acyl-CoA to enter the mitochondrion the carnitine shuttle is used:
1. Acyl-CoA
is
transferred
to
the
hydroxyl
group of carnitine
by carnitine
palmitoyltransferase I, located on the cytosolic faces of the outer and inner mitochondrial
membranes.
2. Acyl-carnitine is shuttled inside by a carnitine-acylcarnitine translocase, as a carnitine is
shuttled outside.
3. Acyl-carnitine is converted back to acyl-CoA by carnitine palmitoyltransferase II, located
on the interior face of the inner mitochondrial membrane. The liberated carnitine is
shuttled back to the cytosol, as an acyl-CoA is shuttled into the matrix.

Beta oxidation, in the mitochondrial matrix, then cuts the long carbon chains of the fatty
acids (in the form of acyl-CoA molecules) into a series of two-carbon (acetate) units,
which, combined with co-enzyme A, form molecules of acetyl CoA, which condense
with oxaloacetate to form citrate at the "beginning" of the citric acid cycle. It is
convenient to think of this reaction as marking the "starting point" of the cycle, as this is
when fuel - acetyl-CoA - is added to the cycle, which will be dissipated as CO2 and H2O
with the release of a substantial quantity of energy captured in the form of ATP, during
the course of each turn of the cycle.
Briefly, the steps in β-oxidation (the initial breakdown of free fatty acids into acetylCoA) are as follows:
1. Dehydrogenation by acyl-CoA dehydrogenase, yielding 1 FADH2
2. Hydration by enoyl-CoA hydratase
3. Dehydrogenation by 3-hydroxyacyl-CoA dehydrogenase, yielding 1 NADH + H+
4. Cleavage by thiolase, yielding 1 acetyl-CoA and a fatty acid that has now been shortened
by 2 carbons (forming a new, shortened acyl-CoA)
This β-oxidation reaction is repeated until the fatty acid has been completely reduced to acetylCoA or, in, the case of fatty acids with odd numbers of carbon atoms, acetyl-CoA and
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
1 molecule of propionyl-CoA per molecule of fatty acid. Each β-oxidative cut of the acyl-CoA
molecule yields 5 ATP molecules.

The acetyl-CoA produced by β-oxidation enters the citric acid cycle in the mitochondrion
by combining with oxaloacetate to form citrate. This results in the complete combustion
of the acetyl-CoA to CO2 and water. The energy released in this process is captured in the
form of 1 GTP and 11 ATP molecules per acetyl-CoA molecule oxidized. This is the fate
of acetyl-CoA wherever β-oxidation of fatty acids occurs, except under certain
circumstances in the liver. In the liver oxaloacetate can be wholly or partially diverted
into the gluconeogenic pathway during fasting, starvation, a low carbohydrate diet,
prolonged strenuous exercise, and in uncontrolled type 1 diabetes mellitus. Under these
circumstances oxaloacetate is hydrogenated to malate which is then removed from the
mitochondrion to be converted into glucose in the cytoplasm of the liver cells, from
where it is released into the blood. In the liver, therefore, oxaloacetate is unavailable for
condensation with acetyl-CoA when significant gluconeogenesis has been stimulated by
low (or absent) insulin and high glucagon concentrations in the blood. Under these
circumstances acetyl-CoA is diverted to the formation of acetoacetate and betahydroxybutyrate. Acetoacetate, beta-hydroxybutyrate, and their spontaneous breakdown
product, acetone, are frequently, but confusingly, known as ketone bodies (as they are not
"bodies" at all, but water-soluble chemical substances). The ketone bodies are released by
the liver into the blood. All cells with mitochondria can take ketone bodies up from the
blood and reconvert them into acetyl-CoA, which can then be used as fuel in their citric
acid cycles, as no other tissue can divert its oxaloacetate into the gluconeogenic pathway
in the way that this can occur in the liver. Unlike free fatty acids, ketone bodies can cross
the blood-brain barrier and are therefore available as fuel for the cells of the central
nervous system, acting as a substitute for glucose, on which these cells normally survive.
The occurrence of high levels of ketone bodies in the blood during starvation, a low
carbohydrate diet, prolonged heavy exercise and uncontrolled type 1 diabetes mellitus is
known as ketosis, and, in its extreme form, in out-of-control type 1 diabetes mellitus, as
ketoacidosis.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
The glycerol released by lipase action is phosphorylated by glycerol kinase in the liver (the only
tissue in which this reaction can occur), and the resulting glycerol 3-phosphate is oxidized to
dihydroxyacetone phosphate. The glycolytic enzyme triose phosphate isomerase converts this
compound to glyceraldehyde 3-phosphate, which is oxidized via glycolysis, or converted to
glucose via gluconeogenesis.
Disorders
Disorders of fatty acid metabolism can be described in terms of, for example,
hypertriglyceridemia (too high level of triglycerides), or other types of hyperlipidemia. These
may be familial or acquired.
Familial types of disorders of fatty acid metabolism are generally classified as inborn errors of
lipid metabolism. These disorders may be described as fatty oxidation disorders or as a lipid
storage disorders, and are any one of several inborn errors of metabolism that result from
enzyme defects affecting the ability of the body to oxidize fatty acids in order to produce energy
within muscles, liver, and other cell types.
Hypertriglyceridemia denotes high (hyper-) blood levels (-emia) of triglycerides, the most
abundant fatty molecule in most organisms. Elevated levels of triglycerides are associated with
atherosclerosis, even in the absence of hypercholesterolemia (high cholesterol levels), and
predispose to cardiovascular disease. Very high triglyceride levels also increase the risk of acute
pancreatitis. Hypertriglyceridemia itself is usually symptomless, although high levels may be
associated with skin lesions known as xanthomas.[1]
The diagnosis is made on blood tests, often performed as part of screening. Once diagnosed,
other blood tests are usually required to determine whether the raised triglyceride level is caused
by other underlying disorders ("secondary hypertriglyceridemia") or whether no such underlying
cause exists ("primary hypertriglyceridaemia"). There is a hereditary predisposition to both
primary and secondary hypertriglyceridemia.[1]
Weight loss and dietary modification may improve hypertriglyceridemia. The decision to treat
hypertriglyceridemia with medication depends on the levels and on the presence of other risk
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
factors for cardiovascular disease. Very high levels that would increase the risk of pancreatitis is
treated with a drug from the fibrate class. Niacin and omega-3 fatty acids as well as drugs from
the statin class may be used in conjunction, with statins being the main drug treatment for
moderate hypertriglyceridemia where reduction of cardiovascular risk is required.[1]
Ketone bodies are three water-soluble molecules (acetoacetate, beta-hydroxybutyrate, and their
spontaneous breakdown product, acetone) that are produced by the liver from fatty acids[1] during
periods of low food intake (fasting), carbohydrate restrictive diets, starvation, prolonged intense
exercise,[2] or in untreated (or inadequately treated) type 1 diabetes mellitus. These ketone bodies
are readily picked up by the extra-hepatic tissues, and converted into acetyl-CoA which then
enters the citric acid cycle and is oxidized in the mitochondria for energy.[3] In the brain, ketone
bodies are also used to make acetyl-CoA into long-chain fatty acids. The latter cannot be
obtained from the blood, because they cannot pass through the blood–brain barrier.
Ketone bodies are produced by the liver under the circumstances listed above (i.e. fasting,
starving, low carbohydrate diets, prolonged exercise and untreated type 1 diabetes mellitus) as a
result of intense gluconeogenesis, which is the production of glucose from non-carbohydrate
sources (not including fatty acids).[1] They are therefore always released into the blood by the
liver together with newly produced glucose, after the liver glycogen stores have been depleted.
(These glycogen stores are depleted after only 24 hours of fasting.)[1]
When two acetyl-CoA molecules lose their -CoAs, (or Co-enzyme A groups) they can form a
(covalent) dimer called acetoacetate. Beta-hydroxybutyrate is a reduced form of acetoacetate, in
which the ketone group is converted into an alcohol (or hydroxyl) group (see illustration on the
right). Both are 4-carbon molecules, that can readily be converted back into acetyl-CoA by most
tissues of the body, with the notable exception of the liver. Acetone is the decarboxylated form
of acetoacetate which cannot be converted back into acetyl-CoA except via detoxification in the
liver where it is converted into lactic acid, which can, in turn, be oxidized into pyruvic acid, and
only then into acetyl-CoA.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
Ketone bodies have a characteristic smell, which can easily be detected in the breath of persons
in ketosis and ketoacidosis. It is often described as fruity or like nail polish remover (which
usually contains acetone or ethyl acetate).
Apart from the three endogenous ketone bodies, acetone, acetoacetic acid, and betahydroxybutyric acid,[4] other ketone bodies like beta-ketopentanoate and beta-hydroxypentanoate
may be created as a result of the metabolism of synthetic triglycerides, such as triheptanoin.
Ketosis and ketoacidosis
In normal individuals, there is a constant production of ketone bodies by the liver and their
utilization by extrahepatic tissues. The concentration of ketone bodies in blood is maintained
around 1 mg/dl. Their excretion in urine is very low and undetectable by routine urine tests
(Rothera's test).
When the rate of synthesis of ketone bodies exceeds the rate of utilization, their concentration in
blood increases; this is known as ketonemia. This is followed by ketonuria – excretion of ketone
bodies in urine. The overall picture of ketonemia and ketonuria is commonly referred as ketosis.
The smell of acetoacetate and/or acetone in breath is a common feature in ketosis.
When a type 1 diabetic suffers a biological stress event (infection, heart attack, or physical
trauma), or fails to administer enough insulin they may enter the pathological state of
hyperglycemic ketoacidosis. Under these circumstances, the low or absent insulin levels in the
blood, combined with the inappropriately high glucagon concentrations,[14] induce the liver to
produce glucose at an inappropriately increased rate, causing acetyl-CoA resulting from the betaoxidation of fatty acids, to be converted into ketones bodies. The resulting very high levels of
ketone bodies lower the pH of the blood plasma which reflexively triggers the kidneys to excrete
a very acid urine. The high levels of glucose and ketones in the blood also spill, passively, into
the urine (the ability of the renal tubules to reabsorb glucose and ketones from the tubular fluid,
being overwhelmed by the high volumes of these substances being filtered into the tubular fluid).
The resulting osmotic diuresis of glucose causes the removal of water and electrolytes from the
blood resulting in potentially fatal dehydration.
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Individuals who follow a low-carbohydrate diet will also develop ketosis. This induced ketosis is
sometimes called nutritional ketosis, but the level of ketone body concentrations are on the order
of 0.5-5 mM whereas the pathological ketoacidosis is 15-25 mM.
Disorders of Lipid Metabolism
Fats (lipids) are an important source of energy for the body. The body’s store of fat is constantly
broken down and reassembled to balance the body’s energy needs with the food available.
Groups of specific enzymes help the body break down and process fats. Certain abnormalities in
these enzymes can lead to the buildup of specific fatty substances that normally would have been
broken down by the enzymes. Over time, accumulations of these substances can be harmful to
many organs of the body. Disorders caused by the accumulation of lipids are called lipidoses.
Other enzyme abnormalities prevent the body from converting fats into energy normally. These
abnormalities are called fatty acid oxidation disorders.
Gaucher’s Disease
Gaucher’s disease is caused by a buildup of glucocerebrosides in tissues. Children who have the
infantile form usually die within a year, but children and adults who develop the disease later in
life may survive for many years.
In Gaucher’s disease, glucocerebrosides, which are a product of fat metabolism, accumulate in
tissues. Gaucher’s disease is the most common lipidosis. The disease is most common among
Ashkenazi (Eastern European) Jews. Gaucher’s disease leads to an enlarged liver and spleen and
a brownish pigmentation of the skin. Accumulations of glucocerebrosides in the eyes cause
yellow spots called pingueculae to appear. Accumulations in the bone marrow can cause pain
and destroy bone.
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Other Rare Hereditary Disorders of Lipid Metabolism
Wolman’s disease results when specific types of cholesterol and glycerides accumulate in
tissues. This disease causes enlargement of the spleen and liver. Calcium deposits in the adrenal
glands cause them to harden, and fatty diarrhea (steatorrhea) also occurs. Infants with Wolman’s
disease usually die by 6 months of age.
Cerebrotendinous xanthomatosis occurs when cholestanol, a product of cholesterol
metabolism, accumulates in tissues. This disease eventually leads to uncoordinated movements,
dementia, cataracts, and fatty growths (xanthomas) on tendons. The disabling symptoms often
appear after age 30. If started early, the drug chenodiol helps prevent progression of the disease,
but it cannot undo any damage already done.
In sitosterolemia, fats from fruits and vegetables accumulate in blood and tissues. The buildup
of fats leads to atherosclerosis, abnormal red blood cells, and xanthomas on tendons. Treatment
consists of reducing the intake of foods that are rich in plant fats, such as vegetable oils, and
taking cholestyramine resin.
In Refsum’s disease, phytanic acid, which is a product of fat metabolism, accumulates in tissues.
A buildup of phytanic acid leads to nerve and retinal damage, spastic movements, and changes in
the bone and skin. Treatment involves avoiding eating green fruits and vegetables that contain
chlorophyll. Plasmapheresis, in which phytanic acid is removed from the blood, may be helpful.
Type 1 , the chronic form of Gaucher’s disease, is the most common. It results in an enlarged
liver and spleen and bone abnormalities. Most commonly diagnosed during adulthood, type 1
Gaucher’s disease may lead to severe liver disease, including increased risk of bleeding from the
stomach and esophagus and liver cancer. Neurologic problems can also occur.
Type 2 , the infantile form, usually causes death in the first year of life. Affected infants have an
enlarged spleen and severe neurologic problems.
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Type 3 , the juvenile form, can begin at any time during childhood. Children with type 3 disease
have an enlarged liver and spleen, bone abnormalities, and slowly progressive neurologic
problems. Children who survive to adolescence may live for many years.
Many people with Gaucher’s disease can be treated with enzyme replacement therapy, in which
enzymes are given by vein, usually every 2 weeks. Enzyme replacement therapy is most
effective for people who do not have nervous system complications.
Tay-Sachs Disease
Tay-Sachs disease is caused by a buildup of gangliosides in the tissues. This disease results in
early death.
In Tay-Sachs disease, gangliosides, which are products of fat metabolism, accumulate in tissues.
The disease is most common among families of Eastern European Jewish origin. At a very early
age, children with this disease become progressively intellectually disabled and appear to have
floppy muscle tone. Spasticity develops and is followed by paralysis, dementia, and blindness.
These children usually die by age 3 or 4. The disease cannot be treated or cured.
Before conception, parents can find out whether they carry the gene that causes the disease.
During pregnancy, Tay-Sachs disease can be identified in the fetus by chorionic villus sampling
or amniocentesis.
Niemann-Pick Disease
Niemann-Pick disease is caused by a buildup of sphingomyelin or cholesterol in the tissues. This
disease causes many neurologic problems.
In Niemann-Pick disease, the deficiency of a specific enzyme results in the accumulation of
sphingomyelin (a product of fat metabolism) or cholesterol. Niemann-Pick disease has several
forms, depending on the severity of the enzyme deficiency, which determines how much
sphingomyelin or cholesterol accumulates. The most severe forms tend to occur in Jewish
people. The milder forms occur in all ethnic groups.
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In the most severe form (type A), children fail to grow normally and have several neurologic
problems. These children usually die by age 3. Children with type B disease develop fatty
growths in the skin, areas of dark pigmentation, and an enlarged liver, spleen, and lymph nodes.
They may be intellectually disabled. Children with type C disease develop symptoms during
childhood, with seizures and neurologic deterioration.
Some forms of Niemann-Pick disease can be diagnosed in the fetus by chorionic villus sampling
or amniocentesis. After birth, the diagnosis can be made by a liver biopsy (removal of a tissue
specimen for examination under a microscope). None of the types of Niemann-Pick disease can
be cured, and children tend to die of infection or progressive dysfunction of the central nervous
system. Currently, some therapies that may slow or halt the progression of symptoms in types B
and C are being studied.
Fabry’s Disease
Fabry’s disease is caused by a buildup of glycolipid in tissues. This disease causes skin growths,
pain in the extremities, poor vision, recurrent episodes of fever, and kidney or heart failure.
In Fabry’s disease, glycolipid, which is a product of fat metabolism, accumulates in tissues.
Because the defective gene for this rare disorder is carried on the X chromosome, the full-blown
disease occurs only in males (see Inheritance Patterns : X-Linked Inheritance). The accumulation
of glycolipid causes noncancerous (benign) skin growths (angiokeratomas) to form on the lower
part of the trunk. The corneas become cloudy, resulting in poor vision. A burning pain may
develop in the arms and legs, and children may have episodes of fever. Children with Fabry’s
disease eventually develop kidney failure and heart disease, although most often, they live into
adulthood. Kidney failure may lead to high blood pressure, which may result in stroke.
Fabry’s disease can be diagnosed in the fetus by chorionic villus sampling or amniocentesis. The
disease cannot be cured or even treated directly, but researchers are investigating a treatment in
which the deficient enzyme is replaced by transfusion. Treatment consists of taking analgesics to
help relieve pain and fever or anticonvulsants. People with kidney failure may need a kidney
transplant.
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CHAPTER 4
Purine and Pyrimidine Metabolism
Overview
One of the important specialized pathways of a number of amino acids is the synthesis of purine
and pyrimidine nucleotides. These nucleotides are important for a number of reasons. Most of
them, not just ATP, are the sources of energy that drive most of our reactions. ATP is the most
commonly used source but GTP is used in protein synthesis as well as a few other reactions.
UTP is the source of energy for activating glucose and galactose. CTP is an energy source in
lipid metabolism. AMP is part of the structure of some of the coenzymes like NAD and
Coenzyme A. And, of course, the nucleotides are part of nucleic acids. Neither the bases nor the
nucleotides are required dietary components.We can both synthesize them de novo and salvage
and reuse those we already have.
Nomenclature
Nitrogen Bases
There are two kinds of nitrogen-containing bases - purines and pyrimidines. Purines consist of a
six-membered and a five-membered nitrogen-containing ring, fused together. Pyridmidines
have only a six-membered nitrogen-containing ring. There are 4 purines and 4 pyrimidines that
are of concern to us.
Purines

Adenine = 6-amino purine

Guanine = 2-amino-6-oxy purine

Hypoxanthine = 6-oxy purine

Xanthine = 2,6-dioxy purine
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Adenine and guanine are found in both DNA and RNA. Hypoxanthine and xanthine are not
incorporated into the nucleic acids as they are being synthesized but are important intermediates
in the synthesis and degradation of the purine nucleotides.
Pyrimidines

Uracil = 2,4-dioxy pyrimidine

Thymine = 2,4-dioxy-5-methyl pyrimidine

Cytosine = 2-oxy-4-amino pyrimidine

Orotic acid = 2,4-dioxy-6-carboxy pyrimidine
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Cytosine is found in both DNA and RNA. Uracil is found only in RNA. Thymine is normally
found in DNA. Sometimes tRNA will contain some thymine as well as uracil.
Nucleosides
If a sugar, either ribose or 2-deoxyribose, is added to a nitrogen base, the resulting compound is
called a nucleoside. Carbon 1 of the sugar is attached to nitrogen 9 of a purine base or to
nitrogen 1 of a pyrimidine base. The names of purine nucleosides end in -osine and the names of
pyrimidine nucleosides end in -idine. The convention is to number the ring atoms of the base
normally and to use l', etc. to distinguish the ring atoms of the sugar. Unless otherwise
specificed, the sugar is assumed to be ribose. To indicate that the sugar is 2'-deoxyribose, a d- is
placed before the name.

Adenosine

Guanosine

Inosine - the base in inosine is hypoxanthine

Uridine

Thymidine

Cytidine
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Nucleotides
Adding one or more phosphates to the sugar portion of a nucleoside results in a nucleotide.
Generally, the phosphate is in ester linkage to carbon 5' of the sugar. If more than one phosphate
is present, they are generally in acid anhydride linkages to each other. If such is the case, no
position designation in the name is required. If the phosphate is in any other position, however,
the position must be designated. For example, 3'-5' cAMP indicates that a phosphate is in ester
linkage to both the 3' and 5' hydroxyl groups of an adenosine molecule and forms a cyclic
structure. 2'-GMP would indicate that a phosphate is in ester linkage to the 2' hydroxyl group of
a guanosine. Some representative names are:

AMP = adenosine monophosphate = adenylic acid

CDP = cytidine diphosphate

dGTP = deoxy guanosine triphosphate

dTTP = deoxy thymidine triphosphate (more commonly designated TTP)

cAMP = 3'-5' cyclic adenosine monophosphate
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Polynucleotides
Nucleotides are joined together by 3'-5' phosphodiester bonds to form polynucleotides.
Polymerization of ribonucleotides will produce an RNA while polymerization of
deoxyribonucleotides leads to DNA.
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Purine Catabolism
The end product of purine catabolism in man is uric acid. Other mammals have the enzyme
urate oxidase and excrete the more soluble allantoin as the end product. Man does not have this
enzyme so urate is the end product for us. Uric acid is formed primarily in the liver and excreted
by the kidney into the urine.
Nucleotides to Bases
Guanine nucleotides are hydrolyzed to the nucleoside guanosine which undergoes
phosphorolysis to guanine and ribose 1-P. Man's intracellular nucleotidases are not very active
toward AMP, however. Rather, AMP is deaminated by the enzyme adenylate (AMP)
deaminase to IMP. In the catobilsm of purine nucleotides, IMP is further degraded by
hydrolysis with nucleotidase to inosine and then phosphorolysis to hypoxanthine.
Adenosine does occur but usually arises from S-Adenosylmethionine during the course of
transmethylation reactions. Adenosine is deaminated to inosine by an adenosine deaminase.
Deficiencies in either adenosine deaminase or in the purine nucleoside phosphorylase lead to
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two different immunodeficiency diseases by mechanisms that are not clearly understood. With
adenosine deaminase deficiency, both T and B-cell immunity is affected. The phosphorylase
deficiency affects the T cells but B cells are normal. Whether or not methylated purines are
catabolized depends upon the location of the methyl group. If the methyl is on an -NH2, it is
removed along with the -NH2 and the core is metabolized in the usual fashion. If the methyl is on
a ring nitrogen, the compound is excreted unchanged in the urine.
Bases to Uric Acid
Both adenine and guanine nucleotides converge at the common intermediate xanthine.
Hypoxanthine, representing the original adenine, is oxidized to xanthine by the enzyme xanthine
oxidase. Guanine is deaminated, with the amino group released as ammonia, to xanthine. If this
process is occurring in tissues other than liver, most of the ammonia will be transported to the
liver as glutamine for ultimate excretion as urea.
Xanthine, like hypoxanthine, is oxidized by oxygen and xanthine oxidase with the production of
hydrogen peroxide. In man, the urate is excreted and the hydrogen peroxide is degraded by
catalase. Xanthine oxidase is present in significant concentration only in liver and intestine. The
pathway to the nucleosides, possibly to the free bases, is present in many tissues.
Gouts and Hyperuricemia
Both undissociated uric acid and the monosodium salt (primary form in blood) are only sparingly
soluble. The limited solubility is not ordinarily a problem in urine unless the urine is very acid or
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has high [Ca2+]. [Urate salts coprecipitate with calcium salts and can form stones in kidney or
bladder.] A very high concentration of urate in the blood leads to a fairly common group of
diseases referred to as gout. The incidence of gout in this country is about 3/1000.
Gout is a group of pathological conditions associated with markedly elevated levels of urate in
the blood (3-7 mg/dl normal). Hyperuricemia is not always symptomatic, but, in certain
individuals, something triggers the deposition of sodium urate crystals in joints and tissues. In
addition to the extreme pain accompanying acute attacks, repeated attacks lead to destruction of
tissues and severe arthritic-like malformations. The term gout should be restricted to
hyperuricemia with the presence of these tophaceous deposits.
Urate in the blood could accumulate either through an overproduction and/or an underexcretion
of uric acid. In gouts caused by an overproduction of uric acid, the defects are in the control
mechanisms governing the production of - not uric acid itself - but of the nucleotide precursors.
The only major control of urate production that we know so far is the availability of
substrates (nucleotides, nucleosides or free bases).
One approach to the treatment of gout is the drug allopurinol, an isomer of hypoxanthine.
Allopurinol is a substrate for xanthine oxidase, but the product binds so tightly that the enzyme is
now unable to oxidized its normal substrate. Uric acid production is diminished and xanthine and
hypoxanthine levels in the blood rise. These are more soluble than urate and are less likely to
deposit as crystals in the joints. Another approach is to stimulate the secretion of urate in the
urine.
Summary
In summary, all, except ring-methylated, purines are deaminated (with the amino group
contributing to the general ammonia pool) and the rings oxidized to uric acid for excretion. Since
the purine ring is excreted intact, no energy benefit accrues to man from these carbons.
Pyrimidine Catabolism
In contrast to purines, pyrimidines undergo ring cleavage and the usual end products of
catabolism are beta-amino acids plus ammonia and carbon dioxide. Pyrimidines from nucleic
acids or the energy pool are acted upon by nucleotidases and pyrimidine nucleoside
phosphorylase to yield the free bases. The 4-amino group of both cytosine and 5-methyl cytosine
is released as ammonia.
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Ring Cleavage
In order for the rings to be cleaved, they must first be reduced by NADPH. Atoms 2 and 3 of
both rings are released as ammonia and carbon dioxide. The rest of the ring is left as a betaamino acid. Beta-amino isobutyrate from thymine or 5-methyl cytosine is largely excreted.
Beta-alanine from cytosine or uracil may either be excreted or incorporated into the brain and
muscle dipeptides, carnosine (his-beta-ala) or anserine (methyl his-beta-ala).
De Novo Synthesis of Purine Nucleotides
We use for purine nucleotides the entire glycine molecule (atoms 4, 5,7), the amino nitrogen of
aspartate (atom 1), amide nitrogen of glutamine (atoms 3, 9), components of the folate-onecarbon pool(atoms 2, 8), carbon dioxide, ribose 5-P from glucose and a great deal of energy in
the form of ATP. In de novo synthesis, IMP is the first nucleotide formed. It is then converted to
either AMP or GMP.
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PRPP
Since the purines are synthesized as the ribonucleotides, (not as the free bases) a necessary
prerequisite is the synthesis of the activated form of ribose 5-phosphate. Ribose 5-phosphate
reacts with ATP to form 5-Phosphoribosyl-1-pyrophosphate (PRPP).
This reaction occurs in many tissues because PRPP has a number of roles purine and pyrimidine
nucleotide synthesis, salvage pathways, NAD and NADP formation. The enzyme is heavily
controlled by a variety of compounds (di- and tri-phosphates, 2,3-DPG), presumably to try to
match the synthesis of PRPP to a need for the products in which it ultimately appears.
Commitment Step
De novo purine nucleotide synthesis occurs actively in the cytosol of the liver where all of the
necessary enzymes are present as a macro-molecular aggregate. The first step is a replacement of
the pyrophosphate of PRPP by the amide group of glutamine. The product of this reaction is 5-
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Phosphoribosylamine. The amine group that has been placed on carbon 1 of the sugar becomes
nitrogen 9 of the ultimate purine ring. This is the commitment and rate-limiting step of the
pathway.
The enzyme is under tight allosteric control by feedback inhibition. Either AMP, GMP, or IMP
alone will inhibit the amidotransferase while AMP + GMP or AMP + IMP together act
synergistically. This is a fine control and probably the major factor in minute by minute
regulation of the enzyme. The nucleotides inhibit the enzyme by causing the small active
molecules to aggregate to larger inactive molecules.
[PRPP] also can play a role in regulating the rate. Normal intracellular concentrations of PRPP
(which can and do fluctuate) are below the KM of the enzyme for PRPP so there is great
potential for increasing the rate of the reaction by increasing the substrate concentration. The
kinetics are sigmoidal. The enzyme is not particularly sensitive to changes in [Gln] (Kinetics are
hyperbolic and [gln] approximates KM). Very high [PRPP] also overcomes the normal
nucleotide feedback inhibition by causing the large, inactive aggregates to dissociate back to
the small active molecules.
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Purine de novo synthesis is a complex, energy-expensive pathway. It should be, and is, carefully
controlled.
Formation of IMP
Once the commitment step has produced the 5-phosphoribosyl amine, the rest of the molecule is
formed by a series of additions to make first the 5- and then the 6-membered ring. (Note: the
numbers given to the atoms are those of the completed purine ring and names, etc. of the
intermediate compounds are not given.) The whole glycine molecule, at the expense of ATP adds
to the amino group to provide what will eventually be atoms 4, 5, and 7 of the purine ring (The
amino group of 5-phosphoribosyl amine becomes nitrogen N of the purine ring.) One more atom
is needed to complete the five-membered ring portion and that is supplied as 5, 10-Methenyl
tetrahydrofolate.
Before ring closure occurs, however, the amide of glutamine adds to carbon 4 to start the sixmembered ring portion (becomes nitrogen 3). This addition requires ATP. Another ATP is
required to join carbon 8 and nitrogen 9 to form the five-membered ring.
The next step is the addition of carbon dioxide (as a carboxyl group) to form carbon 6 of the ring.
The amine group of aspartate adds to the carboxyl group with a subsequent removal of fumarate.
The amino group is now nitrogen 1 of the final ring. This process, which is typical for the use of
the amino group of aspartate, requires ATP. The final atom of the purine ring, carbon 2, is
supplied by 10-Formyl tetrahydrofolate. Ring closure produces the purine nucleotide, IMP.
Note that at least 4 ATPs are required in this part of the process. At no time do we have either a
free base or a nucleotide.
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Formation of AMP and GMP
IMP can then become either AMP or GMP. GMP formation requires that IMP be first oxidized
to XMP using NAD. The oxygen at position 2 is substituted by the amide N of glutamine at the
expense of ATP. Similarly, GTP provides the energy to convert IMP to AMP. The amino group
is provided by aspartate in a mechanism similar to that used in forming nitrogen 1 of the ring.
Removal of the carbons of aspartate as fumarate leaves the nitrigen behind as the 6-amino group
of the adenine ring. The monophosphates are readily converted to the di- and tri-phosphates.
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Control of De Novo Synthesis
Control of purine nucleotide synthesis has two phases. Control of the synthesis as a whole
occurs at the amidotransferase step by nucleotide inhibition and/or [PRPP]. The second phase of
control is involved with maintaining an appropriate balance (not equality) between ATP
and GTP. Each one stimulates the synthesis of the other by providing the energy. Feedback
inhibition also controls the branched portion as GMP inhibits the conversion of IMP to XMP and
AMP inhibits the conversion of IMP to adenylosuccinate.
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De Novo Synthesis of Pyrimidine Nucleotides
Since pyrimidine molecules are simpler than purines, so is their synthesis simpler but is still from
readily available components. Glutamine's amide nitrogen and carbon dioxide provide atoms 2
and 3 or the pyrimidine ring. They do so, however, after first being converted to carbamoyl
phosphate. The other four atoms of the ring are supplied by aspartate. As is true with purine
nucleotides, the sugar phosphate portion of the molecule is supplied by PRPP.
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Carbamoyl Phosphate
Pyrimidine synthesis begins with carbamoyl phosphate synthesized in the cytosol of those
tissues capable of making pyrimidines (highest in spleen, thymus, GItract and testes). This uses a
different enzyme than the one involved in urea synthesis. Carbamoyl phosphate synthetase II
(CPS II) prefers glutamine to free ammonia and has no requirement for N-Acetylglutamate.
Formation of Orotic Acid
Carbamoyl phosphate condenses with aspartate in the presence of aspartate transcarbamylase
to yield N-carbamylaspartate which is then converted to dihydroorotate.
In man, CPSII, asp-transcarbamylase, and dihydroorotase activities are part of a
multifunctional protein.
Oxidation of the ring by a complex, poorly understood enzyme produces the free pyrimidine,
orotic acid. This enzyme is located on the outer face of the inner mitochondrial membrane, in
contrast to the other enzymes which are cytosolic. Note the contrast with purine synthesis in
which a nucleotide is formed first while pyrimidines are first synthesized as the free base.
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Formation of the Nucleotides
Orotic acid is converted to its nucleotide with PRPP. OMP is then converted sequentially - not
in a branched pathway - to the other pyrimidine nucleotides. Decarboxylation of OMP gives
UMP. O-PRT and OMP decarboxylase are also a multifunctional protein. After conversion of
UMP to the triphosphate, the amide of glutamine is added, at the expense of ATP, to yield CTP.
Control
The control of pyrimidine nucleotide synthesis in man is exerted primarily at the level of
cytoplasmic CPS II. UTP inhibits the enzyme, competitively with ATP. PRPP activates it.
Other secondary sites of control also exist (e.g. OMP decarboxylase is inhibited by UMP and
CMP). These are probably not very important under normal circumstances.
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In bacteria, aspartate transcarbamylase is the control enzyme. There is only one carbamoyl
phosphate synthetase in bacteria since they do not have mitochondria. Carbamoyl phosphate,
thus, participates in a branched pathway in these organisms that leads to either pyrimidine
nucleotides or arginine.
Interconversion of Nucleotides
The monophosphates are the forms synthesized de novo although the triphosphates are the most
commonly used forms. But, of course, the three forms are in equilibrium. There are several
enzymes classified as nucleoside monophosphate kinases which catalyze the general
reaction:(= represents a reversible reaction)
Base-monophosphate + ATP = Base-diphosphate + ADP
e.g. Adenylate kinase: AMP + ATP = 2 ADP
There is a different enzyme for GMP, one for pyrimidines and also enzymes that recognize the
deoxy forms.
Similarly, the diphosphates are converted to the triphosphates by nucleoside diphosphate
kinase:
BDP + ATP = BTP + ADP
There may be only one nucleoside diphosphate kinase with broad specificity. One can
legitimately speak of a pool of nucleotides in equilibrium with each other.
Salvage of Bases
Salvaging of purine and pyrimidine bases is an exceedingly important process for most tissues.
There are two distinct pathways possible for salvaging the bases.
Salvaging Purines
The more important of the pathways for salvaging purines uses enzymes called
phosphoribosyltransferases (PRT):
PRTs catalyze the addition of ribose 5-phosphate to the base from PRPP to yield a nucleotide.:
Base + PRPP = Base-ribose-phosphate (BMP) + PPi
We gave already seen one example of this type of enzyme as a normal part of de novo synthesis
of the pyrimidine nucleotides, - O-PRT.
As a salvage process though, we are dealing with purines. There are two enzymes, A-PRT and
HG-PRT. A-PRT is not very important because we generate very little adenine. (Remember that
the catabolism of adenine nucleotides and nucleosides is through inosine). HG-PRT, though, is
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exceptionally important and it is inhibited by both IMP and GMP. This enzyme salvages guanine
directly and adenine indirectly. Remember that AMP is generated primarily from IMP, not from
free adenine.
Lesch-Nyhan Syndrome
HG-PRT is deficient in the disease called Lesch-Nyhan Syndrome, a severe neurological
disorder whose most blatant clinical manifestation is an uncontrollable self-mutilation. LeschNyhan patients have very high blood uric acid levels because of an essentially uncontrolled de
novo synthesis. (It can be as much as 20 times the normal rate). There is a significant increase in
PRPP levels in various cells and an inability to maintain levels of IMP and GMP via salvage
pathways. Both of these factors could lead to an increase in the activity of the amidotransferase.
Salvaging Pyrimidines
A second type of salvage pathway involves two steps and is the major pathway for the
pyrimidines, uracil and thymine.
Base + Ribose 1-phosphate = Nucleoside + Pi (nucleoside phosphorylase)
Nucleoside + ATP - Nucleotide + ADP (nucleoside kinase - irreversible)
There is a uridine phosphorylase and kinase and a deoxythymidine phosphorylase and a
thymidine kinase which can salvage some thymine in the presence of dR 1-P.
Formation of Deoxyribonucleotides
De novo synthesis and most of the salvage pathways involve the ribonucleotides. (Exception is
the small amount of salvage of thymine indicated above.) Deoxyribonucleotides for DNA
synthesis are formed from the ribonucleotide diphosphates (in mammals and E. coli).
A base diphosphate (BDP) is reduced at the 2' position of the ribose portion using the protein,
thioredoxin and the enzyme nucleoside diphosphate reductase. Thioredoxin has two
sulfhydryl groups which are oxidized to a disulfide bond during the process. In order to restore
the thioredoxin to its reduced for so that it can be reused, thioredoxin reductase and NADPH
are required.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
This system is very tightly controlled by a variety of allosteric effectors. dATP is a general
inhibitor for all substrates and ATP an activator. Each substrate then has a specific positive
effector (a BTP or dBTP). The result is a maintenance of an appropriate balance of the
deoxynucleotides for DNA synthesis.
Synthesis of dTMP
DNA synthesis also requires dTMP (dTTP). This is not synthesized in the de novo pathway and
salvage is not adequate to maintain the necessary amount. dTMP is generated from dUMP using
the folate-dependent one-carbon pool.
Since the nucleoside diphosphate reductase is not very active toward UDP, CDP is reduced to
dCDP which is converted to dCMP. This is then deaminated to form dUMP. In the presence of
5,10-Methylene tetrahydrofolate and the enzyme thymidylate synthetase, the carbon group is
both transferred to the pyrimidine ring and further reduced to a methyl group. The other product
is dihydrofolate which is subsequently reduced to the tetrahydrofolate by dihydrofolate
reductase.
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Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr
108
Department of Biochemistry-cum-Clinical Biochemistry S.P. College Sgr