Nucleotides, Vitamins, Cosubstrates, and Coenzymes
Objectives:
I. Define the terms cofactor, activator ion, metalloenzymes, coenzyme, cosubstrate, prosthetic group,
vitamin, apoenzyme, and holoenzyme.
II. Describe the structure, precursor vitamin, if present, biological function, and deficiency state for the
cosubstrates / coenzymes:
A. Adenosine triphosphate (ATP)
B. Guanosine triphosphate (GTP)
C. Uridine Triphosphate (UTP)
D. Cytidine Triphosphate (CTP)
E. Nicotinamide adenine dinucleotide (NAD+
F. Nicotinamide adenine dinucleotide phosphate (NADP+))
G. Flavin mononucleotide (FMN)
H. Flavin adenine dinucleotide (FAD)
I. Coenzyme A (CoA)
J. Pyridoxal phosphate
K. Thiamine pyrophosphate
L. Tetrahydrofolate
M. Biotin
N. Lipoic acid
O. Ascorbic acid
P. Vitamin B12
Background - Review
! ! Some enzymes require nonprotein components to attain full enzymatic activity. These nonprotein prosthetic
groups can be metal ions or small organic molecules. Necessary metal ions are called Cofactors or Essential
Ions. Cofactors tightly bound to the protein form Metalloenzymes. Those that are loosely associated with
the protein are termed Activator Ions. The small organic molecules that are tightly bound or covalently
linked to the protein are called Coenzymes. Cosubstrates are diffusible, they diffuse to and bind to the
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protein during the catalytic cycle and then diffuse away to be used by other enzymes.
The APOENZYME is the protein part of the enzyme devoid of its required cofactor, cosubstrate, or coenzyme.
The HOLOENZYME is the active functional enzyme, the protein and its necessary cofactor, cosubstrate, or
coenzyme.
Coenzymes and Cosubstrates are often the metabolically active form of the vitamins. The word VITAMIN
comes from VITAL AMINE. The first vitamins isolated were amines. Vitamins are small organic molecules
that must be obtained from an outside source. Animals obtain vitamins from their diet and/or from the
bacteria that colonize their gastrointestinal tracts.
There are numerous cosubstrates and coenzymes necessary for the proper functioning of cellular enzymes.
A brief description of the most commonly utilized cosubstrates and coenzymes follows:
The Important Cosubstrates and Coenzymes
Nucleotides & Nucleosides General Considerations
When the term NUCLEOTIDE is mentioned in a biochemistry class, students immediately think of either the
precursors for the nucleic acids (ATP, GTP, CTP, UTP, dTTP) or the main energy carrying molecule of the
cell, ATP. All Nucleotides are composed of / release upon hydrolysis:
• A Nitrogen containing Heterocyclic molecule often called the Base
• An aldopentose sugar - Ribose or 2-Deoxyribose
• One or more phosphate(s)
The heterocyclic base is linked to C-1 of the ribose or deoxyribose by an N-glycosidic bond (more on
glycoside bonds when carbohydrates are examined). The first phosphate is linked to the hydroxyl group of
C-5 of the sugar by a phosphoester bond. High energy phosphoanhydride bonds link the second phosphate
to the first and the third to the second, if a second and or third is present.
NUCLEOSIDES are formed when a heterocyclic base is covalently linked to the sugar. Nucleosides are
converted to NUCLEOTIDES by the addition of a phosphate in phosphoester linkage to the 5´-hydroxyl group
of the sugar.
Nucleotide triphosphates and Deoxynucleotide triphosphates are the precursors for RNA and DNA,
respectively. Nucleotides are also:
• Energy storage and energy transfer molecules
• Cosubstrates in transferase reactions
• Carrier molecules of activated intermediates
• Mediators of hormone action
• Local hormones
ADENOSINE-5´-TRIPHOSPHATE. Adenosine-5´-Triphosphate (ATP) is considered by some the single most
important molecule in the cell. Adenosine Triphosphate is a nucleotide composed of the heterocyclic base
ADENINE covalently linked to carbon 1 of β-D-ribose (more about β-D-ribose in the up coming
carbohydrates section). In ATP the first of three phosphate groups is linked to carbon five of ribose by a
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low energy phosphoester bond. Two additional phosphate groups are covalently linked to the first
phosphate by high energy phosphoanhydride bonds. The phosphate bonded to the ribose is the α phosphate,
the middle phosphate is β, and the terminal one is the γ phosphate. Much of the chemical energy used by
cells is stored in the two high energy phosphoanhydride bonds of ATP. ADENOSINE-5´-DIPHOSPHATE (ADP)
is formed when the phosphoanhydride bond between the γ and β phosphate is hydrolyzed. ADENOSINE-5´MONOPHOSPHATE (AMP) is formed when both high energy phosphoanhydride bonds are hydrolyzed. AMP
is a low energy molecule.
NH 2
O
Adenosine-5´-triphosphate
ATP
N
O
N
O
P
O
O
P
O
O
O
P
O
CH 2
NH 2
N
N
N
N
O
O
O
P
O
N
O
CH 2
N
O
O
OH
OH
OH
OH
Adenosine-5´-monophosphate
AMP
NH 2
N
Adenosine-5´-diphosphate
ADP
O
O
N
O
P
O
O
P
O
CH 2
NH 2
N
N
N
N
N
O
O
CH 2
N
O
O
O
P
OH
OH
O
OH
O
Adenosine-3´,5´-cyclicmonophosphate
Cyclic AMP
cAMP
NH 2
N
N
N
N
CH3
O
N
O
N
N
N
HO
CH2
CH 3
Adenosine
O
Caffeine
OH
CH3
O
N
N
H
N
N
CH 3
O
CH3
Theophylline
OH
ATP
is the energy “currency” of the cell. Energy released by the hydrolysis of the high energy
phosphoanhydride bonds in ATP drive many cellular reactions and cellular functions.
reactions that utilize ATP as a cosubstrate (second substrate) usually require Mg+2 as an activator
ion. The Mg+2 ion salt bridges with the negative charges on the phosphates of ATP.
ATP can donate via transferase reactions phosphoryl (–PO3–2), pyrophosphoryl (–P2O6–3), adenylyl
(AMP) or adenosyl groups (nucleoside).
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methionine is coupled to the adenosyl group to form S-Adenosylmethionine (SAM); SAM is an
activated intermediate involved in many methyl group transferase (methylation) reactions.
3´,5´-cyclic AMP (cAMP), formed from ATP, is a second messenger produced intracellularly in
response to a signal molecule binding to its receptor. Second messengers mediate a wide array of
cellular responses by a variety of different mechanisms.
Finally, the nucleoside adenosine has signal molecule properties. It is a local hormone, an AUTOCOID, that
when synthesized and released by a cell is bound to a receptor on nearby cells and causes:
1.
2.
3.
4.
blood vessel dilation
smooth muscle contraction
neurotransmitter release
increased fat metabolism
For example skeletal muscles undergoing rapid or prolonged muscular contraction releases adenosine
causing the surrounding blood vessels to dilate and deliver more oxygen and nutrients to these tissues.
When stressed cells produce and release adenosine. This molecule in the brain has a general overall
calming effect on the organism and has been implicated in sleep regulation. During periods of prolonged
wakefulness extracellular adenosine concentrations increase in the brain and this increase promotes
sleepiness. CAFFEINE seems to function via the adenosine receptor. It binds to the receptor but does not
stimulate a response or does not stimulate the complete response. It acts as competitive inhibitors toward
adenosine.
O
O
O
P
O
O
O
P
O
NH
N
N
O
O
O
N
Guanosine-5´-triphosphate
GTP
P
O
CH 2
N
N
O
N
NH 2
O
O
O
OH
NH
NH 2
P
O
CH 2
O
O
OH
OH
OH
Guanosine-5´-monophosphate
GMP
O
N
Guanosine-5´-diphosphate
GDP
O
O
P
O
N
O
O
P
O
CH 2
NH
N
O
N
NH 2
O
N
O
OH
O
OH
CH 2
NH
N
NH 2
O
O
P
O
O
OH
Guanosine-3´,5´-cyclicmonophosphate
Cyclic GMP
cGMP
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GUANOSINE-5´-TRIPHOSPHATE Guanosine-5´-Triphosphate (GTP) is a nucleotide containing the
heterocyclic base GUANINE. Like ATP, GTP carries energy in phosphoanhydride bonds. In terms of
cosubstrate/coenzyme activity, GTP is the “energy source” for cellular protein synthesis. GTP also serves
an important role in the cellular response to signal molecules, e.g., hormones. 3´,5´-cyclic GMP (cGMP)
formed from GTP is one of the many second messengers produced intracellularly in response to a signal
molecule binding to its receptor. These second messengers mediate a wide array of cellular responses by a
variety of mechanisms.
URIDINE-5´-TRIPHOSPHATE (UTP) Uridine-5´-Monophosphate (UTP) is a nucleotide with the heterocyclic
base URACIL. UTP is used as a cosubstrate and activator/carrier molecule for monosaccharide isomerization
(epimerization) reactions and for the biosynthesis of disaccharides and polysaccharides.
CYTIDINE-5´-TRIPHOSPHATE (CTP) Cytidine-5´-Monophosphate (CTP) is a nucleotide containing the
heterocyclic base CYTOSINE. CTP is used as a cosubstrate and activator/carrier molecule during the
biosynthesis of lipids (phospholipids and sphingolipids).
O
NH
Uridine-5´-triphosphate
UTP
O
O
P
O
O
O
P
O
N
O
O
P
O
CH 2
O
O
N
Cytidine-5´-triphosphate
CTP
O
OH
NH 2
OH
O
O
P
O
O
O
P
O
N
O
O
P
O
CH 2
O
O
O
OH
OH
Nucleic acids are long linear polymers of nucleotides. When nucleic acids are hydrolyzed, nucleoside
monophosphates result. The monomeric unit of nucleic acids are nucleoside monophosphates. Nucleoside
triphosphates are the precursors for the synthesis nucleic acids. The energy released by the hydrolysis of the
two phosphoanhydride bonds is used to form the 3´,5´-phosphodiester bond between the monomer units and
to “drive” the reaction to completion.
DNA, Deoxyribonucleic acids, contain 2´-deoxyribonucleotides, whereas RNA, Ribonucleic acid, contain
ribonucleotides. The backbone of the linear DNA and RNA polymers is comprised of repeating sugar and
phosphate residues held together by 3´,5´ phosphodiester bonds. The heterocyclic bases on DNA are
Adenine (A), Guanine (G), Cytosine (C), and Thymidine (T). In RNA thymidine is replaced by Uracil (U).
Information of DNA and RNA is carried by the unique linear sequence of bases on the long linear polymer.
At one end of each strand there is a 5´ triphosphate group that is not involved in a phosphodiester bond.
This is the 5´ end of the molecule. At the other end there is a free, unreacted 3´ hydroxyl group. This is the
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3´ end of the molecule
O
NH
O
O
P
O
P
O
O
N
O
P
O
O
O
CH2
O
O
O
N
O
O
O
H3C
O
N
P
O
O
CH2
O
P
O
O
NH2
NH
O
P
O
N
O
O
P
O
CH2
N
O
O
O
O
O
N
OH
RNA
N
P
O
O
CH2
N
O
O
CH2
NH2
O
N
O
P
O
NH
O
O
NH2
N
O
O
O
NH
OH
NH2
N
O
NH2
O
N
OH
N
P
O
O
CH2
N
NH2
O
N
N
O
O
O
N
P
O
O
CH2
O
O
NH2
O
N
O
N
P
O
O
CH2
N
O
O
CH2
N
N
O
NH2
O
N
OH
N
P
O
O
CH2
OH
N
P
O
O
CH2
O
O
N
O
O
NH2
O
O
P
N
N
O
DNA
N
OH
OH
N
NH2
O
N
O
O
N
P
O
O
CH2
O
O
OH
NICOTINAMIDE ADENINE DINUCLEOTIDE (NAD+) and NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE
(NADP+). These coenzymes are composed of an AMP molecule covalently linked to a Nicotinamide
Mononucleotide, hence dinucleotide. The nicotinamide part of the molecule comes from the vitamin
NIACIN. NADP+ differs from NAD+ by the presence of an additional phosphate group on carbon two of the
ribose ring of AMP. These molecules are diffusible cosubstrates that take part in oxidation / reduction
reactions. NAD+ and NADP+ are the oxidized forms of the cosubstrate. When reduced, carbon four of the
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nicotinamide ring accepts a HYDRIDE (H–) ion, a proton and two electrons. NAD+/NADP+ always undergo
two electron oxidations or reductions. NADH and NADPH are the abbreviation for the reduced forms.
Oxidized (NAD+)
Hydride Ion (H–)
Nicotinamide
(Vit - Niacin)
Reduced (NADH)
Ribose
AMP
Nicotinamide adenine dinucleotide
Oxidized (NADP+)
Reduced (NADPH)
Hydride Ion
(H–)
Nicotinamide adenine dinucleotide phosphate
The NAD+/NADH pair participates in oxidative reactions involved in energy production. NAD+ accepts
electrons, is the oxidizing agent, during a particular metabolic reaction, the NADH formed during the first
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reaction it then used to reduce a substrate during a subsequent metabolic reaction. The NADP+/NADPH
pair participates in reductive biosynthetic reactions. NADPH acts as the reducing agent.
If a vitamin is completely lacking or is present in the diet at insufficient quantities a deficiency disease
often results. The effects of a diet lacking a single nutrient were determined using laboratory animals.
The results were then extrapolated to the human animal. Whether a deficiency disease due to the lack of
a single nutrient is possible in the human animal other than induced by a laboratory formulated diet is
unclear. With that said, when Niacin is lacking the disease is called Pellagra. This condition is
characterized by dermatitis on exposed areas; stomatitis; atrophic, sore, magenta tongue; impaired
digestion; diarrhea; and disturbances of the CNS.
FLAVIN MONONUCLEOTIDE (FMN) and FLAVIN ADENINE DINUCLEOTIDE (FAD) {below} are both derived
from the vitamin RIBOFLAVIN (B2). FMN is riboflavin linked to a modified form of the sugar ribose (the
sugar alcohol ribitol) with a phosphate group esterified to the hydroxyl group of carbon five. FAD is FMN
linked to an AMP molecule. FMN and FAD take part in oxidation / reduction reactions. The riboflavin ring
usually undergoes a two electron oxidation or reduction. The flavin ring when reduced accepts a pair of
hydrogen atoms, one at N-5 and the second at N-1 of the flavin ring. The reduced form of these molecules
are FMNH2 or FADH2. This coenzyme can, under certain conditions, undergo one electron oxidations or
reductions. For example, it can accept two electrons (2 H atoms) from a substrate in the first step of the
reaction and then pass these two electrons (H atoms), one at a time to two different electron acceptor
molecules. These molecules are usually covalently linked to their apoenzyme, hence they are considered
coenzymes.
Flavin Ring
(Vit - Riboflavin)
2 Hydrogen Atoms
Ribitol
Flavin mononucleotide
[oxidixed form] (FMN)
Flavin mononucleotide
[reduced form] (FMNH2)
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Flavin Ring
(Vit - Riboflavin)
Ribitol
2 Hydrogen Atoms
AMP
Flavin adenine dinucleotide
[oxidixed form] (FAD)
Flavin adenine dinucleotide
[reduced form] (FADH2)
Riboflavin is not known to be the prime factor in a human deficiency disease. Riboflavin deficiency is
characterized by a magenta tongue, fissuring at the corners of the mouth, seborrheic dermatitis, and
corneal vascularization; symptoms similar to Pellagra. It is unclear whether this deficiency disease is
due to a lack of riboflavin or due to a lack of several water soluble vitamins including niacin.
COENZYME A (abbreviated CoA or CoA-SH) is composed of an ADP molecule, linked to a molecule of
PANTOTHENIC ACID (Vit B3) that is then linked to a molecule of 2-mercaptoethylamine. During the
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biosynthesis of CoA, the amino acid cysteine is amide linked to pantothenic acid and subsequently
decarboxylated to form the 2-mercaptoethylamine. The active end of CoA is the free thiol (–SH) group of
the 2-mercaptoethylamine. CoA carries carboxylic acids in thioester linkage. Since thioesters are high
energy chemical bonds, the carboxylic acids carried by CoA are “activated” to undergo a variety of
metabolic reactions. This coenzyme is a diffusible cosubstrate. No known deficiency states.
Pantothenic Acid
Vitamin B3
2-Mercaptoethylamine
(from Cysteine)
ADP
O
O
C
R
OH
C
R
OH
PYRIDOXAL PHOSPHATE is the biochemically active form of the vitamin PYRIDOXAL (Vit B6). Pyridoxal
phosphate is the prosthetic group for a large number of enzymes that catalyze a wide variety of reactions
involving amino acids. These reactions include isomerization, decarboxylation, side chain elimination, and/
or amino group transfer (transamination). The formation of a Schiff base between the amino group on the
substrate and the carbonyl group on pyridoxal phosphate is the first step in the reactions utilizing this
coenzyme. The deficiency state is characterized by growth failure, dermatitis, edema, hypochromic
anemia, convulsions, and CNS changes.
H
O
O
O
P
Schiff base between
Pyridoxal and
an amino acid
H
C
O
H2
C
O
OH
O
O
C
O
N
C
O
C
H2
C
H
OH
O
N
H
P
R
O
N
H
CH 3
CH 3
Pyridoxal phosphate
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THIAMINE PYROPHOSPHATE (TPP) is the metabolically active form of the vitamin THIAMINE (B1). Carbon
two of the thiazolium ring reacts with and carries molecules containing a ketone functional group. It is a
necessary prosthetic group for enzymes that catalyze decarboxylation reactions of 2-keto (α-keto)
carboxylic acids. It is also a prosthetic group for enzymes that transfer ketone groups between molecules
(Transketolase reactions). The deficiency disease is BERIBERI. It is characterized by weight loss, muscle
weakness, muscle wasting, and peripheral neuritis. Sensory changes, anxiety states, and mental
confusion can occur.
H
NH2
H2
C
S
O
N
N
H3C
N
H3 C
C
H2
H2
C
O
O
P
O
P
O
O
O
Thiamine pyrophosphate
TETRAHYDROFOLATE is the biologically active form of the vitamin FOLIC ACID (FOLATE). The folate
molecule is composed of a conjugated PTERIN RING system coupled to 4-AMINOBENZOIC ACID which is
linked to from one to six GLUTAMATE molecules. Tetrahydrofolate (TH4) is important in one carbon
metabolism. It accepts and donates a variety of one carbon fragments bonded to N-5, N-10, or both. These
one carbon fragments are donated to a variety of acceptor molecules. The one carbon fragments carried by
TH4 are at the oxidation levels of methyl, methylene, methanol, formaldehyde, or formic acid. The
deficiency state is characterized by growth failure, anemia, leukopenia, or pancytopenia.
Aminobenzoic
Acid
Pterin Ring
System
O
H
N
C
C
C
H
N
H
O
H2
C
O
O
H2
C
C
H2
O
C
O
N
H
HN
Glutamate
H2N
N
N
H
Tetrahydrofolate
N10
N5
BIOTIN was originally called vitamin H. Biotin serves as a coenzyme for enzymes that catalyze carboxylgroup-transfer reactions and ATP dependent carboxylation reactions (the addition of CO2). The cofactor is
usually covalently linked to the enzyme by an amide bond to the amino group on a lysine side chain. The
usually substrate for the carboxylation reaction is the bicarbonate ion (HCO3–). Mechanistically, the
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reaction involves at least two steps In the first step bicarbonate ion is activated by reaction with ATP and
covalent linkage to N-1 of the biotin ring system. The lysine side chain tether then moves the activated
biotin-carboxyl complex to a different region of the active site where the carboxyl group is passed to an
acceptor molecule.
O
C
HN
NH
H2
C
S
C
H2
H2
C
C
H2
O
C
O
Biotin
LIPOIC ACID can be synthesized in adequate amounts by mammals, so by the strictest definition it is not a
vitamin. Lipoic acid is 6, 8-DITHIOOCTANOIC ACID. The thiols can exist in a five membered oxidized
disulfide ring system or in a reduced (open) form. When active it is covalently linked to the enzyme by an
amide bond to the side chain of a lysine residue. Lipoic acid is involved in oxidation / reduction reactions
and it carries carboxylic acids to and from active sites in multi-enzyme complexes.
O
O
H2
C
H2
C
C
H2
S
H2
C
C
C
H2
H2
C
C
H2
O
S
SH
Lipoic Acid (oxidized form)
C
C
H2
O
SH
Dihydrolipoic Acid (reduced form)
VITAMIN C or ASCORBIC ACID has been discussed in the context of collagen biosynthesis. It is a necessary
coenzyme for the hydroxylation of proline (Prolyl Hydroxylase) and the hydroxylation of lysine (Lyslyl
Hydroxylase) during collagen biosynthesis. Vitamin C also functions as an anti-oxidant within the cell.
Vitamin C or Ascorbic Acid
The deficiency state is SCURVY. The symptoms of scurvy in adults include spongy gums, loosening of
the teeth, bleeding into the skin and mucous membranes due to increased capillary fragility, and
decreased immunocompetence. Bone development in children is abnormal resulting in bowed legs and/
or stunted growth. With a severe or prolonged deficiency of vitamin C there is decreased wound healing,
osteoporosis, hemorrhaging, and anemia.
VITAMIN B12 or COBALAMINE takes part in only two reactions in humans. It is necessary for the Methionine
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Synthase a transferase reaction which methylates homocysteine to methionine and it is necessary for LMethylmalonyl-CoA Mutase which converts L-methylmalonyl-CoA to succinyl-CoA. Vitamin B12
deficiency results in the accumulation of both homocysteine and L-methylmalonyl-CoA. B12 deficiency
presents with megaloblastic anemia with neurological deterioration caused by progressive demyelination.
Vitamin B12 is widespread in foods of animal origin, especially meats. Since the liver can store up to a six
year supply, deficiencies are rare except in older individuals and in long term vegetarians.
OH
OH
O
N
H 3C
N
N
N
NH 2
H 2N
O
H 2N
H 3C
O
C
H2
C
C
CH 3
CH 2
C
O
CH 2
O
CH 2
C
NH 2
CH 2
H2
C
N
H 2N
Co 3+
N
N
H 3C
CH 3
N
H
H2
C
H 3C
CH 3
CH 2
O
CH 2
CH 2
CH 2
C
C
NH 2
NH
CH 3
NH 2
C
O
O
CH 2
HC
CH 3
O
H 3C
N
O
P
O
OH
H 3C
N
O
OH
H 2C
13
OH
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