Coenzymes, vitamins, trace
elements
Josef Fontana
EC - 41
Overview of the lecture
• Introduction to the topic: cofactors
– Revision of the enzyme structure
– The interrelationship cofactor - vitamin - trace element
• Simple enzymes (proteases)
• Examples of complex enzymes and function of
cofactors in their molecules
– Vitamins as cofactors (decarboxylases,
dehydrogenases)
– Trace elements as cofactors (carbonic anhydrase)
– Cofactors of enzyme groups
• The importance for doctors - deficiency as a
disease / deficiency as a treatment of diseases
Introduction to the topic:
cofactors
Revision of the enzyme
structure
Structure and properties
• most of enzymes are proteins
The figure is found at: http://fig.cox.miami.edu/~cmallery/255/255enz/enzymology.htm (December 2006)
Enzyme structure
• Enzymes are mostly proteins (exception: ribozyme catalytic active RNA).
• Some enzymes in addition to protein component
contain also non-protein component. According to
this we can divide enzymes into:
– Simple enzymes contain only protein (pepsin, trypsin,
ribonuclease).
– Complex enzymes contain protein and non-protein
component = cofactor.
• Cofactor is the non-protein part of the enzyme
molecule. It is necessary for its catalytic function.
Our body can not synthesize them often - therefore,
we eat their precursors – e.g. vitamins.
Introduction to the topic:
cofactors
The interrelationship
cofactor - vitamin - trace
element
Cofactor can be
• 1) metal ion: Zn2+, Mn2+, Mg2+, Fe2+, Cu2+ (trace
elements)
• 2) organic molecule:
– coenzymes are slightly bound to the enzyme, undergo a
chemical change and are released from the enzyme
molecule, they are derivates of vitamins very often:
NAD(P)+, FAD, coenzyme Q, ...
– prosthetic groups are tightly bound to the enzyme and
remain associated with enzyme during the whole
reaction: heme, FAD,…
• Coenzyme + apoenzyme (inactive protein) →
holoenzyme (active enzyme)
The figure is found at:
http://stallion.abac.peachnet.edu/sm/kmccrae/BIOL2050/Ch1-13/JpegArt1-13/05jpeg/05_jpeg_HTML/index.htm (December
2006)
Simple enzymes (proteases)
Proteases - enzymes that
cleave the peptide bond
H
O
R'
H
N
N
N
R
H
O
H
H2O
protease
R'
O
N
OH
R
+
H
N
H2N
O
Bond cleavage requires the presence of water (hydrolysis).
Side chains of amino acids play a key role in the reaction
Simple enzymes
• How looks like the catalytic mechanism of
simple enzymes ? I show you two examples:
• 1) pepsin, which belongs to the aspartate
proteases and
• 2) chymotrypsin, which belongs to the serine
proteases
• Neither of them needs to have cofactor for
the hydrolysis. Both need properly arranged
side chains of amino acids in their active site.
How works pepsin
• For the pepsin catalytic activity are essential two
aspartates (their side chains) in its active site
and water molecule.
• One aspartate attracts proton from water
molecule. Water then attacks the carbonyl group
(that forms a peptide bond).
How works pepsin
• Catalysis involves two conserved
aspartyl residues.
• In the first stage of the reaction, an
aspartate functioning as a general
base (Asp X) extracts a proton from
a water molecule, making it more
nucleophilic.
• This resulting nucleophile then
attacks the electrophilic carbonyl
carbon of the peptide bond targeted
for hydrolysis, forming a tetrahedral
transition state intermediate.
How works pepsin
• A second aspartate (Asp Y) then
facilitates the decomposition of
this tetrahedral intermediate by
donating a proton to the amino
group produced by rupture of the
peptide bond.
• The two different active site
aspartates can act simultaneously
as a general base or as a general
acid because their immediate
environment favors ionization of
one, but not the other.
How works chymotrypsin
• While catalysis by aspartic proteases involves the
direct hydrolytic attack of water on a peptide bond,
catalysis by the serine protease chymotrypsin
involves prior formation of a covalent acyl enzyme
intermediate.
• A highly reactive seryl residue, serine 195,
participates in a charge-relay network with histidine
57 and aspartate 102.
• Far apart in primary structure, in the active site
these residues are within bond-forming distance of
one another. Aligned in the order Asp 102-His 57-Ser
195, they constitute a "charge-relay network" that
functions as a "proton shuttle."
How works chymotrypsin
CH2OH
H
N
CH2
N
CH2-COOH
all these tools come from amino acids
in the protein active site
How works chymotrypsin
• Binding of substrate initiates
proton shifts that in effect
transfer the hydroxyl proton
of Ser 195 to Asp 102.
• The enhanced nucleophilicity
of the seryl oxygen
facilitates its attack on the
carbonyl carbon of the
peptide bond of the
substrate, forming transient
tetrahedral intermediate.
How works chymotrypsin
• The proton on Asp 102 then
shuttles through His 57 to the
amino group liberated when the
peptide bond is cleaved.
• The portion of the original
peptide with a free amino group
then leaves the active site and
is replaced by a water molecule,
yielding an acyl-Ser 195
intermediate (covalent bond
acyl-enzyme).
How works chymotrypsin
• The charge-relay network now
activates the water molecule by
withdrawing a proton through His 57
to Asp 102.
• The resulting hydroxide ion attacks
the acyl-enzyme intermediate,
forming a second tetrahedral
intermediate.
• The charge-relay system donates a
proton to Ser 195 (restoring its
original state), facilitating
breakdown of tetrahedral
intermediate to release the carboxyl
terminal peptide.
Examples of complex enzymes
and function of cofactors in
their molecules
Vitamins as cofactors
(decarboxylases,
dehydrogenases)
Vitamins
• Name derives from the Latin word Vita =
Life
• Vitamins soluble in water:
• Vitamin C
• B complex: B1, B2, B3, B6, folic acid (B9), B12,
niacin (PP), biotin, pantothenoic acid
• Fat-soluble vitamins: Vitamin A, D, E, K
B1 Thiamine
• Active cofactor is thiamine
pyrophosphate - TPP (produced by TPPsynthetase)
• TPP is a cofactor in oxidative
decarboxylation of pyruvate and alphaketoglutarate.
• In addition, TPP is used in transketolase
reactions (pentose phosphate pathway).
Thiamine → TPP
Thiamine pyrophosphate
• The key part of the cofactor molecule is thiazole ring with
its acidic hydrogen.
• Hydrogen is removed by the enzyme, forming carbanion.
Molecule is called ylide (contains anion next to the cation).
• Anion can then react with the carbonyl group in different
molecules (e.g. pyruvate).
• Pyrophosphate works as a handle, which holds the cofactor
in proper place within the enzyme molecule.
Thiamine pyrophosphate
• The key part of the
cofactor molecule is
thiazole ring with
its acidic hydrogen.
• Hydrogen is
removed by the
enzyme, forming
carbanion. Molecule
is called ylide
(contains anion next
to the cation).
Thiamine pyrophosphate
• TPP catalyses the 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).
• It achieves this in four basic steps.
O
Cl
Cl
CH3
Thiamine pyrophosphate
N
O-
N
O
H
• 1) 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.
CH3
H
pyruvate
S
S
ylid
acidic hydrogen
Cl
CH3
Cl
- CO2
CH3
N
N
S
HO
S
O
OH
O-
resonance
Cl
CH3
CH3
N
N
H
+
S
S
OH
O
H
H
O
+
H3C
H
acetaldehyde
ylid
O
Cl
Cl
CH3
Thiamine pyrophosphate
N
O-
N
O
H
• 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.
CH3
H
pyruvate
S
S
ylid
acidic hydrogen
Cl
CH3
Cl
- CO2
CH3
N
N
S
HO
S
O
OH
O-
resonance
Cl
CH3
CH3
N
N
H
+
S
S
OH
O
H
H
O
+
H3C
H
acetaldehyde
ylid
O
Cl
Cl
CH3
Thiamine pyrophosphate
N
• 4) The TPP-substrate
bond is broken, reforming
the TPP ylid and the
substrate carbonyl
(decarboxylated
substrate) is transferred
on the target molecule
(e.g. lipoamide).
O-
N
O
H
• 3) The electrons push
back in the opposite
direction forming a new
bond between the
substrate carbon and
hydrogen proton.
CH3
H
pyruvate
S
S
ylid
acidic hydrogen
Cl
CH3
Cl
- CO2
CH3
N
N
S
HO
S
O
OH
O-
resonance
Cl
CH3
CH3
N
N
H
+
S
S
OH
O
H
H
O
+
H3C
H
acetaldehyde
ylid
thiazolium ring
NH2
H
C
N
N
S
O
H3C
N
H3C
H2
C
H2C
O
O
P
O
P
O-
O-
O-
thiamine pyrophosphate
H
NH2
H3C
C
OH
C
N
N
S
O
H3C
N
H3C
H2C
H2
C
O
P
O-
Hydroxyethyl thiamine pyrophosphate
O
O
P
O-
O-
Lipoic acid
• Lipoic acid is a co-factor found in pyruvate
dehydrogenase and α-ketoglutarate
dehydrogenase and two multienzymes involved
in α-keto acid oxidation.
• Lipoic acid couples acyl group transfer and
electron transfer during oxidation and
decarboxylation of α-ketoacids.
• No evidence exists of a dietary lipoic acid
requirement in humans. Therefore it is not
considered to be a vitamin.
S
SH HS
S
CH
H2C
COOH
C
H2
lipoic acid, reduced form
H
S
CH
H2C
C
H2
COOH
C
H2
lipoic acid, oxidized form
S
CH
H2C
NH
N
C
O
CH
C
O
lipoamide complex (lipoyl-lysine conjugate)
Lipoic acid exists in 2 forms: a closed-ring disulfide form and
an open-chain reduced form; oxidation-reduction cycles interconvert
these 2 species; lipoic acid exists covalently attached in an amide
linkage with lysine residues on enzymes
B2 Riboflavin
• It forms 2 cofactors :
• flavin mononucleotide, FMN
• flavin adenine dinucleotide,
FAD
• Involved in the metabolism
of carbohydrates, fats and
proteins (flavin
dehydrogenases /
flavoproteins).
• Hydrogen carriers in the
respiratory chain
H3C
H2C
OH
H
C
OH
H
C
OH
H
C
OH
H
C
H
N
N
O
N
H3C
N
H
O
RIBOFLAVIN
Riboflavin
H
H3 C
N
N
O
N
H
O
FAD (oxidized form)
N
N
O
N
N
H3 C
H3C
H3C
N
H
hydrogen addition
occurs in 2 steps
H
O
FADH2 (reduced form)
Succinate dehydrogenase
CO2CH2
succinate
C
H
CH2
CO2-
CO2-
succinate
dehydrogenase
FAD
FADH2
H
C
CO2fumarate
Xanthine oxidase
OH
OH
N
N
OH
N
N
N
N
OH
N
N
HO
N
N
H
hypoxanthine
HO
N
N
H
xanthine
H
uric acid
xanthine oxidase
Xanthine oxidase is a flavoprotein which also contains Fe and Mo – trace elements
Examples of complex enzymes
and function of cofactors in
their molecules
Trace elements as cofactors
(carbonic anhydrase)
Trace elements
• Present in human body in amount less than
1mg/kg.
• I, Zn, Cu, Se, Cr, F, Mn, Co, Mo
• Have known biological function (prosthetic
groups).
• Deficiencies are huge problem in developing
countries.
• But also specific risk groups: the homeless,
alcoholics and patients on total parenteral
nutrition (TPN)
Carbonic anhydrase
• Catalyzes the reaction:
• The active site contains zinc.
Carbonic anhydrase
• A zinc prosthetic group
in the enzyme is held by
3 histidine side-chains.
The fourth coordination
position is occupied by
water.
• This causes polarisation
of the hydrogen-oxygen
bond in water, making the
oxygen slightly more
negative, thereby
weakening the bond.
Carbonic anhydrase
• A fourth histidine is placed
close to the substrate of
water and accepts a proton.
This leaves a hydroxide
attached to the zinc.
• The active site also contains
specificity pocket for carbon
dioxide, bringing it close to
the hydroxide group. This
allows the electron rich
hydroxide to attack the
carbon dioxide, forming
bicarbonate.
• Bicarbonate is replaced with
a new water molecule.
Examples of complex enzymes
and function of cofactors in
their molecules
Cofactors of enzyme groups
Cofactors help to catalyze many
reactions
• Cofactors of oxidoreductases: NAD(P)+,
FAD, cytochromes (contain heme), Fe-S
complexes
• Coenzymes carrying C1 radicals:
tetrahydrofolate, vitamin B12, Sadenosylmethionine, biotin (cofactor of
carboxylases)
• Cofactors carrying acyl: lipoic acid (PDH
prosthetic group, α-KGDH) HSCoA,
pyridoxal phosphate (transaminases)
Cofactors of oxidoreductases
NAD+
NADP+
nicotinamide adenine dinucleotide
nicotinamide aden. dinucl. phosphate
(precursor: niacin = nicotinic acid)
H+
FAD
FMN
flavin adenine dinucleotide
flavin mononucleotide
2 H+
(precurzor: riboflavin = vitamin B2)
heme
Fe3+ + e- ↔ Fe2+
⇒
e-
Cofactors of transferases
ATP
GTP
TDP
adenosine triphosphate
guanosine triphosphate
thiamine diphosphate
/ phosphate
/ phosphate
/ C-fragment
PALP
pyridoxal phosphate
/ -NH2
(prekurzor: thiamine = vitamin B1)
(prekurzor: pyridoxine = vitamin B6)
THF
tetrahydrofolate
(prekurzor: folic acid)
/ C1-fragment
CoA
coenzyme A (HS-Co-A) / acyl
PAPS phosphoadenosine phosphosulfate / sulfate
Cofactors of
Lyases:
PALP pyridoxal phosphate (decarboxylases)
Ligases:
ATP adenosine triphosphate
→ acyl-CoA-synthetases
→ aminoacyl-tRNA-synthetases
biotin
= vitamin H (carboxylases)
Coenzymes and prosthetic group
NAD+ ↔ NADH + H+
nicotinamide adenine dinucleotide
coenzyme
FAD ↔ FADH2
flavin adenine dinucleotide
(vit. B2 = riboflavin)
prosthetic gr.
Other examples: coenzyme A, coenzyme Q, tetrahydrofolate, thiamine
diphosphate (vit. B1 = thiamine)
http://web.indstate.edu/thcme/mwking/vitamins.html
Prosthetic groups
Biotin (vit. H)
Heme
• Another example: pyridoxal phosphate (derivate
of vitamin B6)
http://web.indstate.edu/thcme/mwking/vitamins.html
The importance for doctors deficiency as a disease / deficiency
as a treatment of diseases
B9 Folic Acid
• Folic acid is composed of pteridine, P-amino benzoic acid,
and glutamic acid.
• Its derivate is tetrahydrofolate. It can carry one-carbon
fragments (involved in the methylation reactions).
Folate deficiency causes
megaloblastic anemia
• N5, N10-methylene-tetrahydrofolate carries
methyl group into the thymine synthesis
(necessary for DNA synthesis and thus also
for the formation of erythrocytes).
• Deficiency of folic acid affects cells that are
dividing rapidly because they have a large
requirement for thymidine for DNA synthesis.
• Clinically, this affects the bone marrow,
leading to megaloblastic anemia.
Lack of folate can treat tumors
• During the thimine synthesis there are several
changes in the N5, N10-methylene-THF molecule.
• The methylene group is reduced to the methyl group
that is transferred, and tetrahydrofolate is oxidized
to dihydrofolate.
• For further pyrimidine synthesis to occur,
dihydrofolate must be reduced back to
tetrahydrofolate, a reaction catalyzed by
dihydrofolate reductase.
• Dividing cells, which must generate thymidine and
dihydrofolate, thus are especially sensitive to
inhibitors of dihydrofolate reductase such as the
anticancer drug methotrexate.
Lack of folate can treat
infections
• Human body can not produce its own
THF, while pathogenic bacteria have
this ability.
• This is used in the treatment of
bacterial diseases with
sulphonamides.
• They are in fact for bacteria
indistinguishable similar to paminobenzoic acid.
• Bacteria try to incorporate
sulphonamides into THF molecules,
but the result is a dysfunctional
cofactor.
• Sulfonamides are the competitive
inhibitors of enzymes involved in
synthesis of THF.