COENZYMES AND VITAMINS
INTRODUCTORY BIOCHEMISTRY
COENZYMES AND VITAMINS
Introduction
• Some enzymes require cofactors for activity
Essential ions (mostly metal ions)
Coenzymes (organic compounds)
Apoenzyme + Cofactor
(protein only)
(inactive)
Holoenzyme
(active)
2
COENZYMES AND VITAMINS
Introduction
TYPES OF COFACTORS
• Essential ions and coenzymes can be further distinguished
by the strength of interaction with their apoenzymes.
3
ESSENTIAL IONS
Many Enzymes Require Inorganic Ions
• Enzymes requiring metal ions for full activity:
Metal-activated enzymes have an absolute
requirement or are stimulated by metal ions (examples:
K+, Ca2+, Mg2+)
Metalloenzymes contain firmly bound metal ions at
the enzyme active sites (examples: iron, zinc, copper,
cobalt )
4
ESSENTIAL IONS
Many Enzymes Require Inorganic Ions
EXAMPLE: Mechanism of carbonic anhydrase
• Action of carbonic anhydrase, a
metalloenzyme
• Zinc ion promotes the ionization of bound
H2O. Resulting nucleophilic OH- attacks
carbon of CO2
ESSENTIAL IONS
Many Enzymes Require Inorganic Ions
IRON IN METALLOENZYME
• Iron undergoes reversible oxidation and reduction:
Fe3+ + e- (reduced substrate) à Fe2+ + (oxidized substrate)
• Enzyme heme groups and cytochromes
contain iron
• Nonheme iron exists in iron-sulfur clusters
(iron is bound by sulfide ions and S- groups
from cysteines)
• Iron-sulfur clusters can accept only one e- in a
reaction
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COENZYMES
Classification
• There are two classes of coenzymes based on how they interact with
the apoenzyme:
Cosubstrates are altered during the reaction and regenerated by
another enzyme
Prosthetic groups remain bound to the enzyme during the
reaction, and may be covalently or tightly bound to enzyme
• They can also be classified according to their source:
Metabolite coenzymes – synthesized from common metabolites
Vitamin-derived coenzymes – derivatives of vitamins (vitamins
cannot be synthesized by mammals, but must be obtained as
nutrients)
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METABOLLITE COENZYMES
ATP
• A number of nucleosides and nucleotides are coenzymes
• Adenosine triphosphate (ATP) is the most abundant
• ATP is a versatile reactant that can donate its:
1) Phosphoryl group (γ-phosphate)
2) Pyrophosphoryl group (γ,β phosphates)
3) Adenylyl group (AMP)
4) Adenosyl group
8
METABOLLITE COENZYMES
ATP and Other Nucleotide Cosubstrates
• ATP is also a source of other metabolite coenzymes such as Sadenosylmethionine (SAM)
Methionine + ATP à S-Adenosylmethionine + Pi + PPi
• SAM donates methyl groups in many biosynthesis reactions
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METABOLLITE COENZYMES
ATP and Other Nucleotide Cosubstrates
• Methylation reactions that require SAM include methylation of
phospholipids, proteins, DNA, and RNA
• SAM donates the methyl group for the synthesis of the hormone
epinephrine from norepinephrine
• In plants, SAM acts as a precursor of the plant hormone ethylene –
involved in fruit ripening
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METABOLLITE COENZYMES
ATP and Other Nucleotide Cosubstrates
• Nucleotide-sugar
coenzymes are involved in
carbohydrate metabolism
• Most common: uridine
diphosphate glucose
(UDP-glucose)
• Other variants: CDP, GDP,
and ADP
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VITAMIN-DERIVED COENZYMES
Classification
Type
Coenzyme of active form
Function promoted
Thiamine
Thiamine pypophosphate (TPP)
Aldehyde group transfer
Riboflavin
Flavin mononucleotide (FMN)
Flavin adenine dinucleotide (FAD)
H atom (electron) transfer
Nicotinic acid
Nicotinamide adenine dinucleotide (NAD)
Nicotinamide adenine dinucleotide
phosphate (NADP)
H atom (electron) transfer
H atom (electron) transfer
Pantothenic acid
Coenzyme A
Acyl-group transfer
Pyridoxine
Pyridoxal phosphate
Amino group transfer
Biotin
Biocytin
Carboxyl group transfer
Vitamin B12
Coenzyme B12
1,2 shift of H atoms
Lipoic acid
Lipoylline
H atom and acyl group transfer
Ascorbic acid
-----
Hydroxylation
WATER SOLUBLE
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VITAMIN-DERIVED COENZYMES
Classification
Type
Coenzyme of active form
Function promoted
Vitamin A
11-cis-retinal
Visual cycle
Vitamin D
1,25-dihydroxycholecalciferaol
Ca and P metabolism
Vitamin E
-----
Antioxidant
Vitamin K
-----
Prothrombin biosynthesis
FAT SOLUBLE
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VITAMIN-DERIVED COENZYMES
Nutrition
• Vitamins are required for
coenzyme synthesis and
must be obtained from
nutrients
• Animals rely on plants and
microorganisms for vitamin
sources (meat supplies
vitamins also)
• Most vitamins must be
enzymatically transformed
to the coenzyme
Some vitamins and their associated
deficiencies
Vitamin
Disease
Ascorbate (C)
Scurvy
Nicotinic acid (B3)
Pellagra
Riboflavin (B2)
Growth retardation
Pantothenate (B5)
Dermatitis in chickens
Thiamine (B1)
Beriberi
Pyridoxal (B6)
Dermatitis in rats
Biotin
Dermatitis in humans
Folate
Anemia
Cobalamin (B12)
Pernicious anemia
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VITAMIN-DERIVED COENZYMES
Thiamine Pyrophosphate (TPP)
• TPP is a derivative of thiamine (Vit B1)
• TPP is synthesized from thiamine by enzymatic transfer of a
pyrophosphoryl group from ATP
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VITAMIN-DERIVED COENZYMES
Thiamine Pyrophosphate (TPP)
• Reactive center is the thiazolium ring (with a very acidic hydrogen
atom at C-2 position)
• TPP participates in reactions of:
(1) Decarboxylation
(2) Oxidative decarboxylation
(3) Transketolase enzyme reactions
• In these reactions the thiazole ring of TPP serves as a transient
carrier of a covalently bound “active aldehyde” group. Mg2+ is also
required as cofactor
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VITAMIN-DERIVED COENZYMES
Thiamine Pyrophosphate (TPP)
Example: Pyruvate dehydrogenase complex
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VITAMIN-DERIVED COENZYMES
Thiamine Pyrophosphate (TPP)
Example: Mechanism of pyruvate dehydrogenase (1/2)
18
VITAMIN-DERIVED COENZYMES
Thiamine Pyrophosphate (TPP)
Example: Mechanism of pyruvate dehydrogenase (2/2)
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VITAMIN-DERIVED COENZYMES
FAD and FMN
• Flavin adenine dinucleotide
(FAD) and Flavin mononucleotide (FMN) are derived
from riboflavin (Vit B2)
Ribofavin
• Riboflavin is a derivative of
isoalloxazine
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VITAMIN-DERIVED COENZYMES
FAD and FMN
• Flavin coenzymes are
involved in oxidationreduction reactions for
many enzymes
(flavoenzymes or
flavoproteins)
FMN (black) and FAD (black and blue)
• FAD and FMN catalyze one
or two electron transfers
• The oxidized forms are
yellow. On reduction they
undergo bleaching with a
characteristic change in
the absorption spectrum
21
VITAMIN-DERIVED COENZYMES
FAD and FMN
22
VITAMIN-DERIVED COENZYMES
NAD+ and NADP+
• Nicotinic acid (niacin) is precursor of
nicotinamide adenine dinucleotide (NAD+)
and nicotinamide adenine dinucleotide
phosphate (NADP+)
• Lack of niacin causes the disease pellagra in
humans and black tongue in dogs
• Plants and most animals can make nicotinic
acid from other precursors, particularly
tryptophan
• Nicotinic acid is the 3-carboxyl derivative of
pyridine
• The amide of nicotinic acid is an essential
component of NAD+ and NADP+
23
VITAMIN-DERIVED COENZYMES
NAD+ and NADP+
• NAD+ and NADP+ function as coenzymes of a large number of
oxidoreductases collectively known as pyridine-linked
dehydrogenases
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VITAMIN-DERIVED COENZYMES
NAD+ and NADP+
• Oxidation by pyridine nucleotides always occurs two electrons at a
time
• Dehydrogenases transfer a hydride ion (H:-) from a substrate to
pyridine ring C-4 of NAD+ or NADP+
• The net reaction is:
NAD(P)+ + 2e- + 2H+ à NAD(P)H + H+
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VITAMIN-DERIVED COENZYMES
NAD+ and NADP+
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VITAMIN-DERIVED COENZYMES
NAD+ and NADP+
• Reaction of lactate dehydrogenase
• NAD+ is bound first and NADH released last
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VITAMIN-DERIVED COENZYMES
NAD+ and NADP+
Mechanism of lactate dehydrogenase
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VITAMIN-DERIVED COENZYMES
NAD+ and NADP+
Malate dehydrogenase reaction
+
⇋
L-Malate
+
Oxaloacetate
NAD+
NADH.H+
29
VITAMIN-DERIVED COENZYMES
Coenzyme A (CoA or CoA-SH)
• Derived from the vitamin pantothenate (Vit B5), which is an amide of
β-alanine and pantoate
• The reactive centre of CoA is the –SH group
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VITAMIN-DERIVED COENZYMES
Coenzyme A (CoA or CoA-SH)
• CoA functions as a carrier of acyl groups in enzymatic reaction involved in:
1) Fatty acid oxidation
2) Fatty acid synthesis
3) Pyruvate oxidation
4) Biological acetylations
• Acyl groups are covalently attached to the -SH of CoA to form thioesters
31
VITAMIN-DERIVED COENZYMES
Pyridoxal Phosphate (PLP)
• PLP is derived from Vit B6 family of vitamins (deficiencies lead to
dermatitis and disorders of protein metabolism)
• Vitamin B6 is phosphorylated to
form PLP
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• Transamination is the most
frequently encountered PLPdependent reaction catalyzed by
transaminases or
aminotransferases
• In transamination, the α-amino
group of an aa is transferred to
the carbonyl group on an α-keto
acid to produce a new aa
• PLP-dependent enzymes catalyze
amino-group transfer by forming
enzyme-bound PMP, as a
covalent intermediate
• A Schiff’s base results from the
condensation of a primary amine
with an aldehyde or ketone
33
VITAMIN-DERIVED COENZYMES
Pyridoxal Phosphate (PLP)
Mechanism of transaminases
34
VITAMIN-DERIVED COENZYMES
Biotin
• Biotin is required in very small amounts because it is available from
intestinal bacteria
• Avidin (raw egg protein) binds biotin very tightly and may lead to a
biotin deficiency (cooking eggs denatures avidin so it does not bind
biotin)
• Biotin contains fused imidazole and thiophene rings
• Biotin is linked by an amide bond to the ε-amino group of a lysine
residue of the enzyme à biocytin or biotinyllysine is the active form
35
VITAMIN-DERIVED COENZYMES
Biotin
• Biotin (a prosthetic group) enzymes catalyze:
(1) Carboxyl-group transfer reactions
(2) ATP-dependent carboxylation reactions
• Biotin (a prosthetic group) serves as an intermediate carrier of CO2
during the action of certain carboxylating enzymes (such as pyruvate
carboxylase) in the form of a very labile carboxybiotin derivative:
36
VITAMIN-DERIVED COENZYMES
Biotin
Reaction catalyzed by pyruvate carboxylase
Pyruvate + HCO3- + ATP à oxaloacetate + ADP + Pi
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VITAMIN-DERIVED COENZYMES
Tetrahydrofolate (THF)
• Folic acid (Latin folium, leaf), also known as pteroylglutamic acid or
folacin, was first found in spinach leaves and is broadly distributed in
plants. Also found in liver and yeast
Substituted pteridine
(pterin)
ρ-aminobenzoic acid
glutamate
38
VITAMIN-DERIVED COENZYMES
Tetrahydrofolate (THF)
• The coenzyme THF (or FH4) is a folate derivative and where positions
5,6,7,8 of the pterin ring are reduced in two steps (next slide)
• THF contains 5-6 glutamate residues which facilitate binding of the
coenzyme to enzymes
• THF participates in transfers of one carbon units at the oxidation levels
of methanol (CH3OH), formaldehyde (HCHO), formic acid (HCOOH)
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VITAMIN-DERIVED COENZYMES
Tetrahydrofolate (THF)
• Folate is reduced in two NADPH-dependent steps in a reaction catalyzed
by dihydrofolate reductase (DHFR)
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VITAMIN-DERIVED COENZYMES
Tetrahydrofolate (THF)
One-carbon derivatives of THF
•
The derivatives can be
interconverted enzymatically
by the routes shown
•
R represents the benzoyl
polyglutamate portion of
tetrahydrofolate
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VITAMIN-DERIVED COENZYMES
Tetrahydrofolate (THF)
Example of folate cycle
NADH.H+
NAD+
Methyl
transferase
H2O
Glycine
Dihydrofolate
reductase
Serine
Serine
hydroxymethyltransferase
(PLP-dep.)
42
VITAMIN-DERIVED COENZYMES
Lipoamide
• Coenzyme lipoamide is the proteinbound form of lipoic acid
• Although lipoic acid is often described
as a vitamin, animals can synthesize it
• Lipoic acid is an 8-carbon carboxylic acid
with a cyclic disulfide
• It reduced open chain form
dihydrolipoic acid, which has two
sulfhydryl groups at C6 and C8
Lipoic acid
(oxidized form)
Dihydrolipoic acid
• Lipoamide is believed to function as a swinging arm that carries acyl
groups between the active sites in the multienzyme complexes such
as the pyruvate dehydrogenase complex and α-ketoglutarate
dehydrogenase complex
43
VITAMIN-DERIVED COENZYMES
Lipoamide
• Lipoic acid is bound via an amide linkage to the ε-amino group of an
enzyme lysine
• Reactive center of the coenzyme shown in red
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VITAMIN-DERIVED COENZYMES
Cobalamin (Vitamin B12)
Cobalamin has two components:
1.
Corrin ring system (black), which ressembles the porphyrin ring system of
hemoglobin
2.
A ribonuclotide containing
as base 5,6dimethylbenzimidazole
(blue), which is coordinated
with the cobalt of the corrin
ring and is also bound via a
phosphoester linkage to a
side chain of the corrin ring
system.
45
VITAMIN-DERIVED COENZYMES
Cobalamin (Vitamin B12)
• Humans obtain cobalamin (also known as cyanocobalamin) from foods
of animal origin (deficiency leads to pernicious anemia)
• It is required in trace amounts by animals; normal human blood
contains 0.0002 μg/ml
• Neither animals nor plants can synthesize vitamin B12. It is synthesized
by only a few microorganisms
• Pernicious anemia, a potentially fatal disease in which there is a
decrease in the production of blood cells by bone marrow.
• Pernicious anemia can also cause neurological disorders
• Most victims do not secrete a necessary glycoprotein called intrinsic
factor from the stomach mucosa. This protein specifically binds
cobalamin and the complex is absorbed by cells of the small intestine
46
VITAMIN-DERIVED COENZYMES
Cobalamin (Vitamin B12)
Cobalamin coenzymes
• Coenzymes: methylcobalamin, adenosylcobalamin
47
VITAMIN-DERIVED COENZYMES
Cobalamin (Vitamin B12)
Methylcobalamin participates in the transfer of methyl groups
48
VITAMIN-DERIVED COENZYMES
Cobalamin (Vitamin B12)
Adenosylcobalamin-dependent enzymes catalyze intramolecular
rearrangements
Rearrangement in which a
hydrogen atom and a
substituent on an adjacent
carbon atom exchange places
Rearrangement of
methylmalonyl CoA to
succinyl CoA, catalyzed by
methylmalonyl-CoA mutase
49
Vitamin C
Not a Coenzyme
• Vitamin C or ascorbic acid is one of the simplest vitamins in
structure, being a lactone of a sugar acid
• Most animals can synthesize ascorbic acid in the course of
carbohydrate metabolism, but primates (including humans) lack
this ability and should take it in their diet
Ascorbic acid
Dehydroascorbic acid
(oxidized form)
50
Vitamin C
A Reducing Agent
• It is a reducing reagent for hydroxylation of collagen:
Ø It acts as a cofactor in the enzymatic hydroxylation reactions
such as hydroxylation of proline to hydroxyproline
Ø However it is not specific in these reactions and can be
replaced by other reducing agents
51
VITAMIN-DERIVED COENZYMES
Lipid Vitamins
• Four lipid vitamins: A, D, E, K
• All contain rings and long, aliphatic side chains
• All are highly hydrophobic
• The lipid vitamins differ widely in their functions
52
VITAMIN-DERIVED COENZYMES
Lipid Vitamins – Vitamin A (Retinol)
• Vit A is obtained from liver, egg yolks, milk products or β-carotene
from yellow vegetables
• Vit A exists in 3 forms: the alcohol retinol, aldehyde retinal and
retinoic acid
53
VITAMIN-DERIVED COENZYMES
Lipid Vitamins – Vitamin A (Retinol)
• Retinol and retinoic acid are signal compounds that bind to
receptor proteins inside cells; the ligand-receptor complexes
then bind to chromosomes and can regulate gene expression
during cell differentiation
• Retinal is a light-sensitive compound with a role in vision. It is
the prosthetic group of the protein rhodopsin; absorption of a
photon of light by retinal triggers a neural impulse
54
VITAMIN-DERIVED COENZYMES
Lipid Vitamins – Vitamin D
• Vit D3 (cholecalciferol) is formed nonenzymatically in the skin
from the steroid 7-dihydrocholesterol when exposed to sufficient
sunlight
• 1,25-dihydroxycholecalciferol is the active form of vitamin D3,
formed by 2 hydroxylation reactions. The active compounds are
hormones that help regulate Ca2+ utilization in humans
55
VITAMIN-DERIVED COENZYMES
Lipid Vitamins – Vitamin D
• Vit D2 is the additive in fortified milk
• Deficiencies in Vit D causes rickets in children and osteomalacia
in adults. Bones are weak because calcium phosphate does not
properly crystallize on the collagen matrix of bones
56
VITAMIN-DERIVED COENZYMES
Lipid Vitamins – Vitamin E
• Vit E (α-tocopherol) is a reducing reagent that scavenges oxygen
and free radicals
• May prevent damage to fatty acids in membranes
• A deficiency in vit E is rare but may lead to fragile red blood
cells and neurological damage
57
VITAMIN-DERIVED COENZYMES
Lipid Vitamins – Vitamin K
• Vitamin K or (phylloquinone) is lipid vitamin from plants
required for synthesis of blood coagulation proteins
• A coenzyme for mammalian carboxylases that convert
glutamate to γ-carboxyglutamate residues
• Calcium binds to the γ-carboxyGlu residues of these coagulation
proteins which adhere to platelet surfaces
• Vitamin K analogs are sometimes administered to individuals
who suffer from excessive blood clotting. They are competitive
inhibitors in the carboxylation of glutamate residues
58
VITAMIN-DERIVED COENZYMES
Lipid Vitamins – Vitamin K
59