TPP

• TPP – Thiamine Pyrophosphate

• Thiamine pyrophosphate (TPP or ThPP), or thiamine

diphosphate (TDP), or cocarboxylase is
a thiamine (vitamin B1) derivative which is produced by
the enzyme thiamine diphosphokinase.

• Thiamine pyrophosphate is a cofactor that is present in

all living systems, in which it catalyzes several
biochemical reactions.
 SOURCES - TPP
• TPP is synthesized in the cytosol and is required in the
cytosol for the activity of transketolase and in the
mitochondria for the activity of pyruvate-, oxoglutarate-
and branched chain keto acid dehydrogenases.
• To date, the yeast TPP carrier system mitochondrial
transport of TPP
• Drosophila melanogaster have been identified as being
responsible for the mitochondrial transport of TPP.
• It was first discovered as an essential cofactor.
• Food sources of thiamin include whole grains, meat,
and fish.
• Breads, cereals, and infant foods are fortified with
thiamin.
 STRUCTURE - TPP
• Chemically, TPP consists of a pyrimidine ring which is
connected to a thiazole ring, which is in turn connected
to a pyrophospahte group (diphosphate) functional
group.
• The part of TPP molecule that is most commonly involved
in reactions is the thiazole ring, which contains nitrogen
and sulfur.
• Thus, the thiazole ring is the reagent portion of the
molecule.
 MECHANISM - TPP
• In several reactions, including that of pyruvate
dehydrogenase, alpha-ketoglutarate dehydrogenase, and
transketolase, TPP catalyses the reversible
decarboxylation reaction

• Cleavage of a substrate compound at a carbon carbon

bond connecting a carbonyl group to an adjacent reactive
group - usually a carboxylic acid or an alcohol).
 MECHANISM - TPP
• It achieves this in four basic steps
1. The C2 of this ring is capable of acting as an acid by
donating its proton and forming a carbanion.
• Normally, reactions that form carbanions are highly
unfavorable, but the positive charge on the tetravalent
nitrogen just adjacent to the carbanion stabilizes the
negative charge, making the reaction much more
favorable.
• A compound with positive and negative charges on
adjacent atoms is called an ylid or ylide, so sometimes
the carbanion form of TPP is referred to as the ylid form.
• The carbanion of the TPP ylid nucleophilically attacks
the carbonyl group on the substrate. (This forms a single
bond between the TPP and the substrate.)
 MECHANISM - TPP
2. The target bond on the substrate is broken, and its electrons
are pushed towards the TPP.
• This creates a double bond between the substrate carbon
and the TPP carbon and pushes the electrons in the N-C
double bond in TPP entirely onto the nitrogen atom,
reducing it from a positive to neutral form.
3. In what is essentially the reverse of step two, the electrons
push back in the opposite direction forming a new bond
between the substrate carbon and another atom. (In the
case of the decarboxylases, this creates a new carbon-
hydrogen bond.
• In the case of transketolase, this attacks a new substrate
molecule to form a new carbon-carbon bond.)
4. In what is essentially the reverse of step one, the TPP-
substrate bond is broken, reforming the TPP ylid and the
substrate carbonyl.
 REACTIONS - TPP

Pyruvate decarboxylase
Removes carboxyl group from pyruvate and forms
acetaldehyde
MECHANISM - TPP
 SIGNIFICANCE - TPP
• TPP works as a coenzyme in many enzymatic reactions,
such as:
Pyruvate dehydogenase Complex
Pyruvate decarboxylase complex
Alpha ketogluterate dehydrogenase
Branched chain aminoacid dehydrogenase complex
2 hydroxyphytanoyl CoA lysae
Transketolase.
• It serves as a cofactor for enzymes involved in
carbohydrate metabolism, including transketolase, α-
ketoglutarate dehydrogenase, pyruvate dehydrogenase,
and branched chain α-keto acid dehydrogenase.
 DISEASES - TPP
• Deficiency of thiamine can affect the cardiovascular,
nervous, and immune system, as is commonly seen in
wet beriberi, dry beriberi,
• Worldwide it is most widely reported in populations
where polished rice and milled cereals are the primary
food source, and also in patients with chronic alcohol
abuse.
• Dry beriberi presents as symmetrical peripheral
neuropathy while wet beriberi presents with high-output
heart failure.
• Wernicke-Korsakoff syndrome (WKS) can manifest with
CNS symptoms such as gait changes, altered mental
status, and ocular abnormalities
 PLP
• Pyridoxal phosphate (PLP, Pyridoxal 5‘- phosphate, P5P),
the active form of vitamin B6, is a coenzyme in a variety
of enzymatic reactions.
• PLP is required for over 100 different reactions in human
metabolism, primarily in the various amino acid
biosynthetic and degradation pathways.
• PLP participation of allows for decarboxylation of amino
acids, a chemical step which would be highly unlikely
without the coenzyme, and PLP is also required for a very
important class of biochemical transformation called
'transamination', in which the amino group of an amino
acid is transferred to an acceptor molecule.
 SOURCES - PLP
• The richest sources of vitamin B6 include fish, beef liver
and other organ meats, potatoes and other starchy
vegetables, and fruit (other than citrus).
• In the United States, adults obtain most of their
dietary vitamin B6 from fortified cereals, beef, poultry,
starchy vegetables, and some non-citrus fruits.
• Vitamin B6 – Rich in pork, poultry, such as chicken or
turkey, some fish, peanuts, soya beans, wheat gram, oats,
bananas.
STRUCTURE - PLP
 MECHANISM - PLP
• PLP acts as a coenzyme in all transamination reactions,
and in certain decarboxylation, deamination and
recimization reactions of amino acids.
• The aldehyde group of PLP forms a schiff base linkage
with the ε-amino group of a specific lysine group of the
amino transferase enzyme.
• The α-amino group of the amino acid substrate displaces
the ε-amino group of the active-site lysine residue in a
process known as transaldimination.
• The resulting external aldimine can lose a proton, carbon
dioxide, or an amino acid side chain to become a
quinonoid intermediate, which in turn can act as a
nucleophile in several reaction pathways.
 MECHANISM - PLP
• The common catalytic cycle of a PLP dependent enzyme
begins and ends with the coenzyme covalently linked to
the enzyme's active site through an imine linkage
between the aldehyde carbon of PLP and the amine
group of a lysine residue.
• For a PLP dependent enzyme to become active, a PLP
molecule must first enter the active site of an enzyme
and form an imine link to the lysine.
• This state is often referred to as an external aldimine.
MECHANISM - PLP
MECHANISM - PLP
REACTIONS - PLP
 REACTIONS - PLP
• Many other amino acid racemase reactions, however,
require the participation of PLP.
1. Racemization Reaction
2. Metabolism and biosynthesis of serotonin.
3. Amino acid decarboxylase reactions
4. Metabolism and biosynthesis of GABA (γ-aminobutyric
acid).
5. Metabolizm of ornithine
 DISEASES - PLP
• Inborn errors leading to PLP deficiency can result in Vitamin B6
responsive epilepsy.
• Epilepsy is a central nervous system (neurological) disorder in
which brain activity becomes abnormal, causing seizures or
periods of unusual behavior, sensations, and sometimes loss of
awareness.
• Anyone can develop epilepsy. Epilepsy affects both males and
females of all races, ethnic backgrounds and ages.
• Two types of epilepsy related to PLP deficiency are pyridoxine
dependent epilepsy and pyridoxal phosphate dependent
epilepsy.
• Pyridoxine dependent epilepsy is mainly caused by mutations in
the ALDH7A1 gene.
• Pyridoxal phosphate dependent epilepsy is mainly caused by
mutations in the PNPO gene. These conditions are mainly seen in
infants, beginning soon after birth or in some cases before birth.
 COENZYME A
• Coenzyme A (CoA, SHCoA, CoASH) is a coenzyme A,
notable for its role in the synthesis and oxidation of fatty
acids, and the oxidation of pyruvate in the citric acid
cycle.
• All genomes sequenced to date encode enzymes that use
coenzyme A as a substrate, and around 4% of cellular
enzymes use it (or a thioester) as a substrate.
• In its acetyl form, coenzyme A is a highly versatile
molecule, serving metabolic functions in both the
anabolic and catabolic pathways.
• Acetyl-CoA is utilised in the post translational regulation
and allosteric regulation of pyruvate dehydrogenase and
carboxylase to maintain and support the partition of
pyruvate synthesis and degradation.
 SOURCES – COENZYME A
• Coenzyme A is naturally synthesized from pantothenate
(vitamin B5), which is found in food such as meat,
vegetables, cereal grains, legumes, eggs, and milk.
• In humans and most living organisms, pantothenate is an
essential vitamin that has a variety of functions.
• In some plants and bacteria, including Escherichia coli,
pantothenate can be synthesised de novo and is
therefore not considered essential.
• These bacteria synthesize pantothenate from the amino
acid aspartate and a metabolite in valine biosynthesis.
 STRUCTURE – COENZYME A

• In humans, CoA biosynthesis requires cysteine,

pantothenate (vitamin B5), and Adenosine tri phosphate
(ATP).
• In all living organisms, coenzyme A is synthesized in a
five-step process that requires four molecules of ATP,
pantothenate and cysteine
STRUCTURE – COENZYME A
 MECHANISM -COENZYME A
• CoA serves as an essential cofactor for some 4% of known
enzymes, including at least 100 enzymes involved in
intermediary metabolism.
• In these reactions CoA forms high-energy thio ester
bonds with carboxylic acids, the most important of which
is acetic acid, which can come from the metabolism of
fatty acids, amino acids, or carbohydrates.
• CoA functions widely in metabolism in reactions involving
either the carboxyl group (e.g., formation of
acetylcholine, acetylated amino sugars, acetylated
sulfonamides) or the methyl group (e.g., condensation
with oxaloacetate to yield citrate) of an acyl CoA.
• Acetyl CoA, the active acetate, group has many metabolic
uses.
 MECHANISM - COENZYME A
• CoA is placed such that the sulfhydryl group is directed
toward the active-site cysteine and within the intimate
region of the active-site cysteine, the CoA binding sites.
• Each structure could accommodate the acetyl group
adjacent to the active-site cysteine.
With carboxylic acids it can form thioesters

• These thioesters are of great importance in biochemical

metabolism since they can be attacked by electrophiles
(including other acyl–CoA molecules and CO2) to form
addition compounds, and by nucleophiles (including
water) to displace the –SCoA group.
 MECHANISM - COENZYME A
• Acetyl coenzyme A is a source of an acetyl group toward
biological nucleophiles; it is an acetyl transfer agent.
 SIGNIFICANCE - COENZYME A
• Coenzyme A is available from various chemical suppliers
as the free acid and lithium and sodium salts.
• The free acid of coenzyme A is detectably unstable, with
around 5% degradation observed after 6 months when
stored at −20°C, and near complete degradation after 1
month at 37 °C.
• The lithium and sodium salts of CoA are more stable,
with negligible degradation noted over several months at
various temperatures.
• Aqueous solutions of coenzyme A are unstable above
pH 8, with 31% of activity lost after 24 hours at 25 °C and
pH 8.
 SIGNIFICANCE - COENZYME A
• CoA stock solutions are relatively stable when frozen at
pH 2–6.
• The major route of CoA activity loss is likely the air
oxidation of CoA to CoA disulfides.
• CoA mixed disulfides, such as CoA-S–S-glutathione, are
commonly noted contaminants in commercial
preparations of CoA.
• Free CoA can be regenerated from CoA disulfide and
mixed CoA disulfides with reducing agents such as
dithiothreitol or 2-mercaptoethanol.
 DISEASES - COENZYME A
• Vitamin B5 deficiency is rare, but may include symptoms
such as fatigue, insomnia, depression, irritability,
vomiting, stomach pains, burning feet, and upper
respiratory infections.
• Pantothenic acid deficiency in humans has been induced
experimentally by co-administering a pantothenic acid
antagonist (omega-methylpantothenic acid) and a
pantothenic acid-deficient diet.
• Participants in this experiment complained of headache,
fatigue, insomnia, intestinal disturbances, and numbness
and tingling of their hands and feet ("burning feet"
syndrome).
• These symptoms could be related to perturbations in CoA
and lipid metabolism.
 LIPOIC ACID
• Lipoic acid (LA), also known as α-lipoic acid and alpha
lipoic acid (ALA) and thioctic acid is an organo sulphur
compound derived from octanoic acid.
• ALA is made in animals normally, and is essential for
aerobic metabolism.
• It is also manufactured and is available as dietary
supplement.
• Lipoic acid is an essential cofactor for the oxidative
decarboxylations of α-keto acids where, linked to the ε-
amino group of a lysine residue of the enzyme
dihydrolipoyl transacetylase, it is one of several
prosthetic groups in the multi-enzyme, lactate
dehydrogenase complex.
 SOURCES – LIPOIC ACID
• Alpha lipoic acid is a natural protective antioxidant.
• Many foods have alpha-lipoic acid in very low amounts.
• They include spinach, broccoli, yams, potatoes, yeast,
tomatoes, Brussels sprouts, carrots, beets, and rice bran.
Red meat - particularly organ meat - is also a source of
alpha-lipoic acid.
• The potential biochemical and therapeutic actions of oral
LA intake can only be appreciated with an understanding
of its bioavailability, tissue accumulation and metabolic
fate.
• Cells maintain active systems to transport, utilize, and
excrete nonprotein-bound LA.
• Typical dietary sources of LA are muscle meats, heart,
kidney, and liver, and to a lesser degree, fruits and
vegetables.
 STRUCTURE - LIPOIC ACID
• Lipoic acid (LA), also known as α-lipoic acid and alpha
lipoic acid (ALA) and thioctic acid is an organo sulfur
compound derived from octanoic acid.
• LA contains two sulfur atoms (at C6 and C8) connected by
a disulphide bond and is thus considered to be oxidized
although either sulfur atom can exist in higher oxidation
states.
• The carbon atom at C6 is chiral (non-superposable mirror
image) and the molecule exists as two enantiomers (R)-
(+)-lipoic acid (RLA) and (S)-(-)-lipoic acid (SLA) and as a
racemic mixture (R/S)-lipoic acid (R/S-LA).
• LA appears physically as a yellow solid and structurally
contains a terminal carboxylic acid and a terminal
dithiolane ring.
STRUCTURE - LIPOIC ACID
 MECHANISM – LIPOIC ACID
• Naturally occurring lipoic acid is known to play a
fundamental role in metabolism, serving as a cofactor in
enzyme complexes, which function at strategic points in
carbohydrate metabolism, citric-acid cycle, and amino-
acid catabolism.
• Lipoate is the conjugate base of lipoic acid, and the most
prevalent form of LA under physiologic conditions.
• Most endogenously produced RLA is not "free" because
octanoic acid, the precursor to RLA, is bound to the
enzyme complexes prior to enzymatic insertion of the
sulfur atoms.
• As a cofactor, RLA is covalently attached by an amide
bond to a terminal lysine residue of the enzyme's lipoyl
domains.
 MECHANISM – LIPOIC ACID
• Only the (R)-(+)-enantiomer (RLA) exists in nature and is
essential for aerobic metabolism because RLA is an
essential cofactor of many enzyme complexes.
• Lipoylated enzymes have lipoic acid attached to them
covalently.
MECHANISM – LIPOIC ACID
 REACTIONS – LIPOIC ACID
• Lipoic acid is cofactor for at least five enzymes systems.
Two of these are in the citric acid cycle through which
many organisms turn nutrients into energy.
• The lipoyl group transfers acyl groups
1. Pyruvate dehydrogenase complex
2. α keto dehydrogenase complex
3. Branched chain oxoacid dehydrogenase complex
(BCDH)
4. 2-Oxoadipate dehydrogenase complex
5. Acetoin dehydrogenase complex
 DISEASES – LIPOIC ACID
• Lipoic acid (LA), is an essential cofactor for mitochondria
enzymes, with some biological functions, such as
antioxidant and anti-inflammatory effects has been
interested in diabetic neuropathy and
neurodegenerative diseases.
• Lipoic acid deficiency has been described in rare cases of
inherited mutations in the lipoic acid biosynthetic
pathway.
• Lipoic acid synthetase deficiency is a rare
neurometabolic disease characterized by a neonatal
onset of seizures (often intractable), muscular hypotonia,
feeding difficulties (poor sucking and/or swallowing) and
mild to severe psychomotor delay, associated with
nonketotic hyperglycinemia.
 BIOTIN
• Biotin also called vitamin H (the H represents Haar und
Haut, German words for Hair and Skin), vitamin
B7 or vitamin B8 (in many countries like France, where
vitamin B7 is used for inositol) is a water soluble B
vitamin.
• It is involved in a wide range of metabolic processes, both
in humans and in other organisms, primarily related to
the utilization of fats, carbohydrates, and amino acids.
• Biotin is an important component of enzymes involved in
metabolizing fats and carbohydrates, influencing cell
growth, and affecting amino acids involved in protein
synthesis.
 BIOTIN
• Biotin assists in various metabolic reactions involving the
transfer of carbon di oxide.
• It may also be helpful in maintaining a steady blood
sugar level.
• Biotin is often recommended as a dietary supplement for
strengthening hair and nails, though scientific data
supporting these outcomes are weak.
• Nevertheless, biotin is found in many cosmetics and
health products for the hair and skin.
 SOURCES – BIOTIN
• The amounts needed are small, and a wide range of foods
contain biotin.
• The Food and Drug Administration (FDA) has established
daily recommended allowance for adults at 30 μg/day.
• Intestinal Bacteria synthesize biotin, but biotin which is
synthesized endogenously by flora of the intestine is not
reabsorbed in the colon, but it is stored as protein-bound
biotin in the bacteria of the intestine and thus not available
to cover the biotin requirements of the human organism.
• The normal biotin blood plasma levels in healthy
individuals range from 200 to 1200 ng/L, with an optimum
level of 400-500 ng/L in younger adults and children.
• Independent from the cause, a biotin deficiency always
exists when the plasma biotin level is below 100 ng/L.
 STRUCTURE - BIOTIN
• Biotin is composed of a ureido ring fused with a tetra
hydro thiopene ring.
• The ureido ring acts as the carbon dioxide carrier in
carboxylation reactions.
• A valeric acid substituent is attached to one of the carbon
atoms of the tetra hydro thiophene ring.
• Biotin has an unusual structure, with two rings fused
together via one of their sides.
• The two rings are ureido and tetra hydro thiohene
moieties.
• Biotin is a heterocyclic, S-containing mono carboxyliic
acid.
 STRUCTURE - BIOTIN
• Biotin is a coenzyme for multiple carboxylase enzymes,
which are involved in the digestion of carbohydrates,
synthesis of fatty acids, and gluconeogenesis.
• Biotin is also required for the catabolism and utilization
of the three branched chain amino acids – Leucine,
isoleucine, and valine.
 MECHANISM - BIOTIN
• Biotin serves as a covalently bound coenzyme for acetyl-
CoA carboxylases 1 and 2, pyruvate carboxylase,
propionyl-CoA carboxylase, and 3-methylcrotonyl-CoA
carboxylase.
• The attachment of biotin to the ε-amino group of a
specific lysine residue in the specific biotin dependent
carboxylase enzyme.
• It is linked to the enzymes by an amide bond between
the amino group of a specific lysyl residue in the active
centre of the respective apocarboxylase and its valeric
acid side chain.
• Biotin dependent carboxylases mediates the
carboxylation reaction.
• Carboxylation and decarboxylation processes are the
main reactions in which biotin is involved.
 MECHANISM - BIOTIN
• Catalysis by all biotin dependent enzymes involves two
half-reactions.
• Biotin dependent enzymes can be divided into three
different classes depending upon whether they function.
1. Class I : Carboxylases
2. Class II : Decarboxylases
3. Class III : Transcarboxylases
• In most cases, biotin dependent enzymes catalyze the
fixation of CO2 and thus belong to the Class I family.
• The general reactions catalyzed by the Class I enzymes
catalytic mechanisms proceed through carboxyphosphate
intermediates.
 MECHANISM - BIOTIN
• It is still not entirely clear as to whether
carboxyphosphate decomposes into CO2 and Pi followed
by reaction of CO2 with biotin or whether the carboxylate
group is transferred directly from carboxyphosphate to
biotin.
• One intriguing proposal suggests that the first half
reaction involves substrate assisted catalysis
whereby Pi serves as the general base required to
abstract the N1 proton from biotin to generate a biotin
enolate - like intermediate.
• Indeed, the nucleophilicity of biotin would be greatly
increased by such an abstraction event.
MECHANISM - BIOTIN
 MECHANISM - BIOTIN
The enolate structural form of the Biotin cofactor
 REACTIONS - BIOTIN
• D-(+)-Biotin is a cofactor responsible for carbondioxide
transfer in several carboxylase enzymes.
1. Acetyl CoA carboxylase – α
2. Acetyl CoA carboxylase – β
3. Methy Corotonyl carboxylase
4. Pyruvate carboxylase
• Biotin is important in fatty acid synthesis, branched-
chain amino acid catabolism, and gluconeogenesis.
 DISEASES - BIOTIN
• Biotin deficiency can be caused by inadequate dietary
intake or inheritance of one or more inborn genetic
disorders that affect biotin metabolism.
• Subclinical deficiency can cause mild symptoms, such as
hair thinning, brittle fingernails, or skin rash, typically on
the face.
• Neonatal screening for biotinidase deficiency have been
screened, obscuring the true prevalence of the disorder.
• A number of rare metabolic disorder exist in which an
individual's metabolism of biotin is abnormal, such as
deficiency in the holocarboxylase syhthetase enzyme
which covalently links biotin onto the carboxylase, where
the biotin acts as a cofactor.
 DISEASES - BIOTIN
• Biotin deficiency typically occurs from the absence of the
vitamin in the diet, particularly in breast feeding
mothers.
• Daily consumption of raw egg whites for several months
may result in biotin deficiency, due to their avidin
content.
• Deficiency can be addressed with nutritional
supplementation.
• Deficiency symptoms of Biotin include - Brittle and thin
fingernails, Hair loss (alopecia), Conjunctivitis, Dermatitis
- in the form of a scaly, red rash around the eyes, nose,
mouth, and genital area.
 DISEASES - BIOTIN
• Neurological symptoms in adults, such as depression,
lethargy, hallucination, and numbness and tingling of the
extremities when the biotin levels are low.
• The neurological and psychological symptoms can occur with
only mild deficiencies.
• Dermatitis, conjunctivitis, and hair loss will generally occur
only when deficiency becomes more severe.
• Individuals with hereditary disorders of biotin deficiency have
evidence of impaired immune system function, including
increased susceptibility to bacterial and fungal infections.
• Pregnant women tend to have a higher risk of biotin
deficiency.
• Nearly half of pregnant women have abnormal increases of 3-
hydroxyisovaleric acid, which reflects the reduced status of
biotin