King Saud University
College of Science
Department of
Biochemistry
Disclaimer
• The texts, tables and images contained in this course presentation
are not my own, they can be found on:
– References supplied
– Atlases or
– The web
Part 3
Coenzymes-Dependent Enzyme
Mechanism
Professor A. S. Alhomida
1
2
Thiamine
3
Thiamine
Pyrimidine
Thiazole
Thiamine contains two heterocyclic rings, primidine and thiazole
participate in the formation of carbanion-TPP
4
• Thiamin is derived from a substituted pyrimidine and a thiazole
which are coupled by a methylene bridge. Thiamin is rapidly
converted to its active form,
thiamin pyrophosphate, TPP, in the brain and liver by a
specific enzymes, thiamin diphosphotransferase. TPP is
necessary as a cofactor for the pyruvate and a-ketoglutarate
dehydrogenase catalyzed reactions as well as the
transketolase catalyzed reactions of the pentose phosphate
pathway. A deficiency in thiamin intake leads to a severely
reduced capacity of cells to generate energy as a result of its
role in these reactions.
The dietary requirement for thiamin is proportional to the caloric
intake of the diet and ranges from 1.0 - 1.5 mg/day for normal
adults. If the carbohydrate content of the diet is excessive then
an in thiamin intake will be required.
5
Conversion of Thiamine into
Coenzyme Form (TPP)
CH3
CH3
N
N
N
H2C
NH2
N
TPP synthetase
H2C
H
N
N
ATP
S
AMP
NH2
H
S
O
H3C
CH2CH2OH
Thiamine
H3C
CH2CH2O
O
P O P
O
O
O
Thiamine pyrophosphate (TPP)
6
Structure of TPP, Cont’d
• 3-D structure
7
Structure of TPP, Cont’d
8
Thiamin Biosynthesis
• The thiamin thiazole in Bacillus subtilis is
biosynthesized from 1-deoxy-D-xylulose-5phosphate (1, DXP), glycine imine 11 and a
sulfur carrier protein thiocarboxylate (4, ThiS–
COS–) as outlined
• In this mechanism, DXP forms an imine with
lysine 96 of the thiazole synthase (ThiG),
which then tautomerizes to 3
9
Thiamin Biosynthesis, Cont’d
• Addition of ThiS-thiocarboxylate 4, followed
by an acyl shift and loss of water gives 7
• Tautomerization of 7 followed by elimination
of ThiS–COO– 10 gives 9 which then adds to
the glycine imine 11, formed by the oxidation
of glycine, to give 12. Transimination followed
by decarboxylation completes the thiazole
formation
10
Thiamin Biosynthesis, Cont’d
• This mechanism is supported by isotope
exchange experiments, intermediate trapping,
the demonstration of oxygen transfer from
DXP to ThiS–COO– and the structural
characterization of thiazole synthase (ThiG)
complexed to the sulfur carrier protein
• This thiazole biosynthesis is different from
any of the characterized chemical or
biochemical routes to the thiazole heterocycle
11
Thiamin Biosynthesis, Cont’d
• A related oxygen-sensitive thiazole
biosynthesis in Escherichia coli that uses
tyrosine instead of glycine has been
reconstituted but not yet mechanistically
characterized
12
Thiamin Biosynthesis, Cont’d
13
TPP and TCA Cycle
• The mechanism is
identical for both the
conversion of pyruvate
to acetyl CoA and the
conversion of a-KG to
succinyl CoA
• In the reaction, the
proton on C-2 of TPP
dissociates to give a
carbanion
14
TPP and TCA Cycle, Cont’d
• Nucleophilic addition by the carbanion to the
carbonyl group of the a-keto acid (i. e.
pyruvate or a-KG) followed by protonation
forms an activated a-hydroxyacid
• The hydroxy acid then undergoes
decarboxylation
• The positively charged nitrogen of TPP
serves as a critical electron sink during the
decarboxylation step and contributes to the
resonance stabilization of the hydroxyalkylTPP decarboxylation product
15
TPP and TCA Cycle, Cont’d
• The hydroxyalkyl group is transferred by
other proteins in the complex to CoA to
produce acetyl CoA from pyruvate or succinyl
CoA from a-KG
16
TPP and Pentose Phosphate Pathway
(PPP)
• PPP harvests energy
from fuel molecules and
stores it in the form of
NADPH
• NADPH is an important
electron donor in
reductive biosynthesis
• PPP also produces 5carbon sugars such as
ribose which is used in
the synthesis of DNA
and RNA
17
TPP and PPP, Cont’d
• TPP is the coenzyme for the transketolase
• Transketolase transfers a 2-carbon unit from
an a-ketose
• 2-carbon unit from the 5-carbon a-ketose
xylulose 5-phosphate is transferred to the 4carbon aldose erythrose 4-phosphate to
make the 6 carbon a-ketose fructose 6phosphate
• GAP results from the 3-carbon fragment that
is cleaved from xylulose 5-phosphate
18
TPP and PPP, Cont’d
• Carbanion at C-2 of TPP is first produced that
attacks the carbonyl carbon of the a-ketose to
give an addition product
• After deprotonation of OH- group an aldose
(in this case GAP) is released and an
activated glycoaldehyde bound to TPP is
produced
• The thiazole nitrogen serves as electron
sink in the reaction and contributes to
resonance stabilization of the resulting
product (activated glycoadehyde)
19
TPP and PPP, Cont’d
• This glycoaldehyde is said to be activated
because it is also a carbanion and readily
undergoes nucleophilic addition to the
carbonyl group of an aldose (erythrose 4phosphate here)
• Following another deprotonation the nascent
a-ketose is released from TPP
20
Neurological Function of TPP
• It is evident from the neurological disorders
caused by thiamine deficiency that this
vitamin plays a vital role in nerve function
• It is unclear, however, just what that role is
• Thiamine is found in both the nerves and
brain
• The concentration of thiamine in the brain
seems to be resistant to changes dietary
concentration
21
Neurological Function of TPP
• Electrical or chemical (e.g., acetylcholine)
stimulation of nerves results in the release of
thiamine monophosphate (TMP) and free
thiamine into the medium with accompanying
decrease of cellular TPP and thiamine
triphosphate (TTP)
• This observation suggest that thiamine has a
role in the nervous system independent of its
coenzyme roles
• One theory is that TTP is involved with nerve
impulses via the Na+ and K+ gradient
22
Thiamine Deficient Diseases
(Beri-beri)
• Thiamine deficiency usually causes weight
loss, cardiac abnormalities, and
neuromuscular disorders
• The classic thiamine deficiency syndrome in
humans is beri-beri (sometimes called Kakke)
• Thiamine is abundant in whole grains, usually
in the scutellum (the thin covering of the
starchy interior endosperm), but is scarce in
the endosperm
23
Thiamine Deficient Diseases
(Beri-beri), Cont’d
• Unfortunately beri-beri is still common in parts
of southeast Asia where polished rice is a
staple and thiamine enrichment programs are
not fully in place
• Beri-beri is characterized by anorexia (loss of
appetite) with subsequent weight loss,
enlargement of the heart, and neuromuscular
symptoms such as paresthesia (spontaneous
sensations, such as itching, burning, etc),
muscle weakness, lassitude (weariness,
general weakness), and foot and wrist droop
24
Thiamine Deficient Diseases
(Beri-beri), Cont’d
• There are three main types of beri-beri:
(1) Dry (also neuritic, paraplegic, and pernicious) beri-beri;
(2) Wet (also edematous or cardiac) beri-beri;
(3) Infantile (also acute) beri-beri
• Dry beri-beri usually inflicts older adults and
affects mainly the peripheral nerves with
• It is characterized by atrophy (wasting away)
and peripheral neuritis (inflammation of
nerves) of the legs and paraplegia (paralysis
of the lower extremities)
25
Thiamine Deficient Diseases
(Beri-beri), Cont’d
• In contrast wet beri-beri displays substantial
cardiac involvement especially tachycardia
(rapid heart beat) in addition to peripheral
neuropathy
• Edema progresses from the feet upwards to
the heart causing congestive heart failure in
severe cases
• Infantile beri-beri is usually seen in breastfeeding infants whose mothers are thiamine
deficient (but not necessarily showing signs
of beri-beri)
26
Thiamine Deficient Diseases
(Beri-beri), Cont’d
• These infants are usually anoretic and often
have trouble keeping the milk down
• Once the disease begins it moves rapidly
causing heart failure in a matter of hours
27
28
Wet Beri-Beri
29
30
TPP and Wernicke-Korsakoff
Syndrome
• It is the thiamine deficient disease seen most
often in the Western hemisphere
• It mainly affects alcoholics due to three
reasons:
(1) Diets of alcoholics are usually poor
(2) Diets rich in carbohydrates (e.g., alcohol or rice)
increase the metabolic demands of thiamine
(3) Alcohol inhibits intestinal ATPase which is
involved in the uptake of thiamine
31
TPP and Wernicke-Korsakoff
Syndrome
• Two observations suggest a genetic
involvement with Wernicke-Korsakoff
Syndrome:
(1) It is much higher in among Europeans than nonEuropeans
(2) Transketolase from these patients binds TPP 10
time less strongly than normal transketolase
32
Decarboxylatoion Reactions
33
Decarboxylatoion Reactions
• Decarboxlation of carboxylic acid leads to the
formation of CO2 and a carbanion
• CO2 is a stable molecule, whereas the
carbaion is a high-energy molecule that
cannot exit for long under the biochemical
conditions
• The main barrier to decarboxylation is the
formation of the carbanion
• The decarboxylation will be facilitated when a
mechanism exists to stabilize the carbanion
produced by decarboxylation
34
Decarboxylation Reaction, Cont’d
R1
R2
O
C
C
R3
Carboxylic acid
O
R2
R1
C
C
+
O
R3
O
CO2
(stable)
Carbanion
(unstable)
35
Decarboxylatoion Reactions, Cont’d
• How can this be accomplished?
• If carbanion is adjacent to an electrondeficient group such as the carbonly group in
a ketone, ester, aldehyde or carboxlyic acid
• It will be stabilized by delocalization of the
electron pair
36
Decarboxylatoion Reactions, Cont’d
• b-Keto acids readily undego decarboxylation,
whereas the carboxylic acid that have no
carbnoly group in the b-position are stable to
decarboxylation under physiological
conditions
• Molecules such as acetic acid, or butyric acid
undergo decarboxylation only under extreme
conditions such as fusion with solid NaOH
37
Decarboxylatoion Reactions, Cont’d
Electron sink
O
H3 C
C
H
C
O
O
H3 C
C
H
b-Ketoacid
O
C
O
H
C
H3 C
C
H
C
+
C
H
H
Carbanion
O
O
Enolate ion
Carbanion stabilization by
delocalization of the
electron pair
38
Decarboxylatoion Reactions, Cont’d
No Electron sink
H3 C
H
H
C
C
H
H
O
O
H3 C
C
O
C
O
H
C
+
C
H
O
Not b-ketoacid
39
Decarboxylatoion Reactions, Cont’d
• How can a decarboxylation reaction be
catalyzed?
• Decarboxylation of a b-keto acid entails the
formation of an enolate ion that is still quite
unstable in neutral pH
• Any interaction with an enzyme that stabilizes
the negative charge will be helpful in the
catalyzing decarboxylation
40
Decarboxylatoion Reactions, Cont’d
• An enzyme-bound enolate can be stabilized by
a positive charged entity such as the proton of
an acidic group or the positive charge of metal
ion placed near the carbonly oxygen
• Stabilization of the enolate lowers the
activation energy for the reaction and
increases the rate
41
Stabilization of Enolate at Active Site
by Acid
General acid donates
hydrogen bond to the bcarbonly group of a bketo acid
General acid donates a H+ to
the enolate anion resulting an
enol intermediate
B
B
H
O
H3 C
C
H
H
C
H
b-Keto acid
O
O
O
H
H3 C
C
O
C
C
C
H
O
Enol intermediate
42
Stabilization of Enolate at Active Site
by Metal Ion
Metal ion polarizes
hydrogen the b-carbonly
group of a b-keto acid
via coordination bond
Metal ion stabilizes the enolate
anion via an electrostatic bond
M2+
O
H3 C
C
M2+
H
C
H
b-Keto acid
O
O
O
H
H3 C
C
O
C
C
C
H
O
Enolate intermediate
43
Decarboxylatoion Reactions, Cont’d
• The enol intermediate is much more stable
than the enolate and it is the intermediate in
enzymatic reaction rather than the enolate
• Conversion of the b-carbonly group into a
protonated imine also facilitates the
decarboxylation
• The pH of an imine is near 7, so that under
biochemical conditions the imine-nitrogen can
be positively charged and acts as a very
effective electron sink
44
Stabilization of Imine
Protonated nitrogen of
imine
R1
H3 C
R1
H
N
C
H
C
H
Electron sink
H
N
O
H3 C
C
O
b-iminium ion carboxylic
acid
C
R1
O
N
H
C
H
H3 C
H
C
C
H
H
Carbanion imine
Enamine
45
+
C
O
Decarboxylatoion Reactions, Cont’d
• Decaboxylation of protonated imine, (bcationic imine,b-iminium ion) leads to the
formation of an enamine
• Enamine is a lower-energy intermediate than
an enolate
• b-Iminium ion nitrogen carries full positive
charge comparing with b-carbonyl group
(partially positive charge)
• b-Iminium ion facilitates decarboxylation
even more effectively than does a b-carbonly
group
46
Decarboxylatoion Reactions, Cont’d
• b-Iminium ion facilitates decarboxylation
even more effectively than does a b-carbonly
group
• If the keto group of a b-ketoacid is converted
into a protonated imine, the rate of
decarboxylation will be greatly enhanced
• As example of enzymatic decarboxylation via
forming imine intermediate:
– Acetoacetate decarboxylase
47
Decarboxylatoion Reactions, Cont’d
• The enzymes catalyze the
dehydrogenations and decarboxylations of
b-hydroxy acids do NOT form imines before
decarboxylation
• They require a divalent cation to facilitate the
decarboxylation through coordination with the
b-carbonly group via providing positive
charge to help stabilize the carbanion
intermediate resulting from decarboxylation
48
Decarboxylatoion Reactions, Cont’d
• Example of enzymes catalyze b-hydroxy
acids:
– Malic enzyme
– Isocitrate dehydrogenase
– 6-phosphogluconate dehydrogenase
49
Decarboxylation of a-Keto Acid
50
Decarboxylation of a-keto Acid
• The decarboxylation of a-keto acids occurs
frequently in biological systems
• It is not obvious that a-keto acids should
decarboxlyate readily, because
decaroxylation of these acids would NOT
produce a stabilized carbanion
• These acids undergo a chemical modification
before decarboxylation, which converts them
into structures resembling b-keto acids
51
Decarboxylation of a-keto Acid, Cont’d
• This chemical modification is facilitated by
TPP
• How does TPP function in decarboxylation of
a-keto acids?
• TPP can undergo a variety of chemical
reactions
• It contains a thiazolium ring can easily be
deprotonated and forms a Zwitter-ion which
reacts as a nucleophile through the carbanion
intermediate
52
Biochemical Functions of TPP
53
Biochemical Functions of TPP
• Reaction with the a-keto acid generates a
heterocyclic enol and carbon dioxide
• Although the enol is relatively stable it retains
its activity because it has lost fully aromatic
character
• The enol reacts as a nucleophile with acarbonyl compounds
54
Biochemical Functions of TPP
BH+
a-Keto acid
TPP
TPP carbanion
55
Biochemical Functions of TPP, Cont’d
• Transfer of the acetyl group restores the
aromatic thiazolium system
• Note that the acetyl group is formally
transferred as an anion with its negative
charge on the carbon of the carbonyl group
• This is reversed polarity and constitutes an
Umpolung
56
Biochemical Functions of TPP, Cont’d
• Reaction with the a-keto acid generates a
heterocyclic enol and carbon dioxide
• Although the enol is relatively stable it retains
its activity because it has lost fully aromatic
character
• In TPP the OH group of thiamine is replaced
by a diphosphate ester group
57
Biochemical Functions of TPP, Cont’d
Electron sink
TPP carbanion
a-Keto acid
HE-TPP
Heterocycle enol
58
Biochemical Functions of TPP, Cont’d
• The reaction site of TPP is C-2 of the thiazole
ring
• Thiazolium ring is responsible for the
enzymatic catalysis carried out by TPP due to
chemical prosperities:
(1) The acidity of the proton attached to C-2
(2) The presence of a C = N (double bond) that can
act as an electron sink for decarboxylation
59
Biochemical Functions of TPP, Cont’d
• The proton on this carbon is rather
acidic because of the adjacent positively
charged quaternary nitrogen atom which
electrostatically stabilizes the carbanion
intermediate formed when the proton
dissociates
• The pKa value of C-2 is near 10 (dipolar
carbanion)
60
Biochemical Functions of TPP, Cont’d
• TPP-C-2 carbanion appears unusually stable
due to electrostatic interaction with the
cationic nitrogen and also d-p orbital overlap
of the negative charge with the adjacent
sulfur atom
• TPP-carbanion has an efficient electron sink
in form of b-iminium ion in b-relation to the
carboxylate group to be decarboxylated as
CO2
61
Biochemical Functions of TPP, Cont’d
• This intermediate provides a low-energy path
to facilitate the decarboxylation reaction of aketo acid
• When this proton dissociates a carbanion is
formed which readily undergoes nucleophilic
addition to a-carbonyl groups
• The carbanion readily adds to carbonly
groups, and the thiazolium ring acts as
electron sink that stabilizes the negative
charge that is transferred to the ring
62
R` N
H
H
H
S
CH3
Thiazolium
R` N
R
CH3
O
N
R` N
R
Oxazolium
H
R
CH3
Imidazolium
63
Comparison Studies
• C-2 oxazolium is more acidic and the oxygen has no
d orbitals, however, it is not catalyst
• Because C-2 is too stable to add weak electrophilies
and unreactive at neutral pH
• C-2 imidazolium is very slow to generate carbanion
intermediate
• Both oxazolium and imidazolium ions are
thermodynamic stable at pH 7 and are not suitable
for conezyme function as thiazolium ion
• The thiazolium ion is the only cone of the three that Is
suitable on thermodynamic and kinetic grounds
•
64
Biochemical Functions of TPP, Cont’d
• TPP is a coenzyme for two types of reactions:
• (1) Decarboxylation
– (1) Nonoxidative decarboxylation
• Yeast pyruvate decarboxylase
– (2) Oxidative decarboxylation
• a-keto acid dehydrogenases
• (2) Transketolaction
– Transketolases
65
TPP-Dependent Enzymes
O
O
O
TPP, RCHO
TPP
H
a-Keto acid
Acetaldehyde
TPP,
FAD,
O2
O
O
Acetic acid
R
COO
TPP,
lipoamide,
CoASH,
NADH,
FAD
OH
a-Hydroxyacetyl
O
SCo A
Acetyl-CoA
66
Biochemical Functions of TPP, Cont’d
• Both of which cleave a C-C bond adjacent to
a carbonyl group releasing either carbon
dioxide or an aldehyde
• The resulting product is then transferred to an
acceptor molecule
• a-Keto acid dehydrogenases decarboxylate
a-keto acids
• The decarboxylation product is then
transferred to CoA
67
Biochemical Functions of TPP, Cont’d
• Transketolases cleaves the C-C bond
adjacent to the carbonyl group of an aketosugar to give an activated glycoaldehyde
• The glycoaldehyde is then combined with an
aldose to give a new ketose
• All known TPP dependent enzymes also
require a divalent cation, commonly Mg2+
68
Mechanism of Pyruvate
Dehydrogenase (PDH)
Complex
69
Reaction of PDH Complex
70
Reaction of PDH Complex, Cont’d
71
Structure of PDH Complex
• The transacetylase core
(E2) is shown in red, the
pyruvate
dehydrogenase (E1) in
yellow, and the
dihydrolipoyl
dehydrogenase (E3) in
green
72
Structure of Transacelylase
• Each red ball
represents a trimer of
three E2 subunits
• Each subunit consists
of three domains:
(1) lipoamide-binding domain
(2) Small domain for
interaction with E3
(3) Large transacetylase
catalytic domain
• All three subunits of the
transacetylase are
shown in red
73
Structure of PDH Complex
• The PDH complex is comprised of multiple
copies of three separate enzymes:
E1: Pyruvate dehydrogenase (or decarboxylase) (2030 copies)
E2: Dihydrolipoyl transacetylase (60 copies)
E3: Dihydrolipoyl dehydrogenase (6 copies)
74
Structure of PDH Complex, Cont’d
75
Structure of PDH Complex, Cont’d
• The complex also requires 5 different
coenzymes:
(1) TPP
(2) CoA
(3) NAD+
(4) FAD+
(5) Lipoamide
• TPP, lipoamide and FAD+ are tightly bound to
enzymes of the complex whereas the CoA
and NAD+ are employed as carriers of the
products of PDH complex activity
76
The coenzymes and Prosthetic
Groups of PDH Complex
Coenzyme
Location
Function
TPP
Bound to E1
Decarboxylates Pyr,
yielding HE-TPP
carbanion
Lipoate
Covalently linked to
Lys on E2 (lipoamide)
Accepts HE carbanion
from TPP as an acetyl
group
CoA
Coenzyme for E2
Accepts the acetyl
group from acetyldihdrolipoamide
FAD
Bound to E3
Reduced by
dihdrolipoamide
NAD+
Coenzyme for E3
Reduced by FADH2
77
Structure of PDH Complex, Cont’d
• PDH complex is a noncovalent assembly of
three different enzymes operating in concert
to catalyze successive steps in the
conversion of pyruvate to acetyl-CoA
• The active sites of all three enzymes are not
far removed from one another, and the
product of the first enzyme is passed directly
to the second enzyme and so on, without
diffusion of substrates and products through
the solution
78
Lipoic acid
• Lipoic acid is a coenzyme found in PDH
complex and a-KGDH complex, two
multienzymes involved in a-keto acid
oxidation
• Lipoic acid functions to:
– Couple acyl group transfer
– Electron transfer during oxidation and
decarboxylation of a-ketoacids
• No evidence exists of a dietary lipoic acid
requirement in humans; therefore it is not
considered a vitamin
79
Structure of Lipoamide
S
• Lipoamide includes a
dithiol that undergoes
oxidation/ reduction
• It acts as a carrier and
an redox agent
CH2
CH2
S
lipoic acid
CH
O
CH2 CH2 CH2 CH2 C
NH
lysine
NH (CH2)4 CH
lipoamide
C
O
2e + 2H+
HS
CH2
HS
CH
CH2
NH
O
CH2 CH2 CH2 CH2 C
NH (CH2)4 CH
dihydrolipoamide
C
80
O
Structure of Lipoamide, Cont’d
1. The carboxyl at the
end of lipoic acid's
hydrocarbon chain
forms an amide bond
to the side-chain
amino group of a
lysine residue of E2
yielding lipoamide
S
CH2
CH2
S
CH
lipoic acid
O
CH2 CH2 CH2 CH2 C
NH
lysine
NH (CH2)4 CH
lipoamide
C
O
2e + 2H+
HS
CH2
CH2
HS
CH
NH
O
CH2 CH2 CH2 CH2 C
NH (CH2)4 CH
C
81
O
Structure of Lipoamide, Cont’d
2. A long flexible arm,
including hydrocarbon
chains of lipoate and
the lysine R-group,
links each lipoamide
dithiol group to one of
2 lipoate-binding
domains of each E2
S
CH2
CH2
S
CH
lipoic acid
O
CH2 CH2 CH2 CH2 C
NH
lysine
NH (CH2)4 CH
lipoamide
C
O
2e + 2H+
HS
CH2
CH2
HS
CH
NH
O
CH2 CH2 CH2 CH2 C
NH (CH2)4 CH
C
82
O
Structure of Lipoamide, Cont’d
3. Lipoate-binding
domains are
themselves part of a
flexible strand of E2
that extends out from
the core of the
complex
S
CH2
CH2
S
CH
lipoic acid
O
CH2 CH2 CH2 CH2 C
NH
lysine
NH (CH2)4 CH
lipoamide
C
O
2e + 2H+
HS
CH2
CH2
HS
CH
NH
O
CH2 CH2 CH2 CH2 C
NH (CH2)4 CH
C
83
O
Structure of Lipoamide, Cont’d
4. The long flexible
attachment allows
lipoamide functional
groups to swing
between E2 active
sites in the core of the
complex and active
sites of E1 and E3 in
the outer shell
S
CH2
CH2
S
CH
lipoic acid
O
CH2 CH2 CH2 CH2 C
NH
lysine
NH (CH2)4 CH
lipoamide
C
O
2e + 2H+
HS
CH2
HS
CH
CH2
NH
O
CH2 CH2 CH2 CH2 C
NH (CH2)4 CH
C
84
O
Structure of Lipoamide, Cont’d
5. E3 binding protein
that binds E3 to E2
also has attached
lipoamide that can
exchange of reducing
equivalents with
lipoamide on E2
S
CH2
CH2
S
CH
lipoic acid
O
CH2 CH2 CH2 CH2 C
NH
lysine
NH (CH2)4 CH
lipoamide
C
O
2e + 2H+
HS
CH2
HS
CH
CH2
NH
O
CH2 CH2 CH2 CH2 C
NH (CH2)4 CH
C
85
O
Structure of Lipoamide, Cont’d
6. Organic arsenicals
are potent inhibitors of
lipoamide-containing
enzymes such as
Pyruvate
Dehydrogenase
H2O
HS
R'
As
O
S
R'
+
As
HS
S
R
R
7. These highly toxic
compounds react with
“vicinal” dithiols such
as the functional group
of lipoamide
86
Mechanism of PDH Complex
87
Formation of TPP-carbanion
H
N
H
H
B:
CH2
H3C
N
CH3
BH+ O
C
Glu
N
N
H
H
S
CH2
R
H3C
B:
N
N
CH3
H
O
C
O
O
Glu
88
S
R
Formation of TPP-carbanion, Cont’d
Electron sink to stabilize the
negative charge
H
N
H
CH2
H3C
N
N
CH3
S
R
O
C
O
Glu
89
Mechanism of PDH Complex
CH3
R1
N
S
R2
Pyruvate
decarboxylase
TPP carbanion
C
CH3
C
O
C
O
BH
O
Pyruvate
90
Mechanism of PDH Complex
• The mechanism is identical for both the
conversion of pyruvate to acetyl CoA and the
conversion of a-KG to succinyl CoA
• In the reaction, the proton on C-2 of TPP
dissociates to give a carbanion
• Nucleophilic addition by the carbanion to the
carbonyl group of the a-keto acid (i. e.
pyruvate or a-KG) followed by protonation
forms an activated a-hydroxy acid
91
Mechanism of PDH complex
• The first step of this reaction, decarboxylation
of pyruvate and transfer of the acetyl group to
lipoamide, depends on accumulation of
negative charge on the carbonyl carbon of
pyruvate
• This is facilitated by the quaternary nitrogen
on the thiazolium group of TPP
92
Decarboxylation step
CH3
R1
N
R1
CH3
N
C
C
OH
Enz
C
O
S
R2
CH3
CH3
O
Tetrahedral intermediate
C
C
OH
Enz
C
O
S
R2
O
Transition state
93
Delocalization of electrons into
iminium electron sink
R1
CH3
N
..
S
CO2
R2
CH3
C
C
CH3
S
R2
Electrophile
R1
CH3
N
OH
Enz
Nucleophile
Dipolar
C
OH
C
Enz
Carbanion of HETPP
Resonance form of hydroxyethyl-TPP
94
S
S
Enz
Electron sink to stabilize
the negative charge
CH3
Dihydrolipoamide
R1
CH3
N
C
S
R2
C
OH
Enz
Hydroxyethyl-TPP
S
S
BH
Enz
Oxidized (dihydrolipoamide(
95
Mechanism of PDH complex, Cont’d
• The cationic imine nitrogen plays three
distinct and important roles in TPP-catalyzed
reactions:
(1) It provides electrostatic stabilization of the
carbanion formed upon removal of the C-2 proton
(The sp2 hybridization and the availability of
vacant d orbitals on the adjacent sulfur probably
also facilitate proton removal at C-2)
(2) TPP nucleophilically attack on pyruvate leads to
decarboxylation
(3) TPP cationic imine nitrogen can act as an
effective electron sink to stabilize the negative
charge that must develop on the carbon that has
been attacked
96
Mechanism of PDH Complex
• The hydroxy acid then undergoes
decarboxylation
• The positively charged nitrogen of TPP
serves as a critical electron sink during the
decarboxylation step and contributes to the
resonance stabilization of the hydroxyalkylTPP decarboxylation product
• The hydroxyalkyl group is transferred by
other proteins in the complex to CoA to
produce acetyl CoA from pyruvate or succinyl
CoA from a-KG
97
Mechanism of PDH complex, Cont’d
• This stabilization takes place by resonance
interaction through the double bond to the
nitrogen atom
• This resonance-stabilized intermediate can
be protonated to give hydroxyethyl-TPP
• This well-characterized intermediate was
once thought to be so unstable that it could
not be synthesized or isolated
98
Mechanism of PDH complex, Cont’d
• However, its synthesis and isolation are
actually routine (In fact, a substantial amount
of the TPP in living things exists as the
hydroxyethyl form)
• The reaction of hydroxyethyl-TPP with the
oxidized form of lipoamide yields the highenergy thiolester of reduced lipoamide and
results in oxidation of the hydroxyl-carbon of
the two-carbon substrate unit
99
B:
Oxidation and
transferring step
R1
CH3
CH3
N
S
H
C C O
R2
Enz
S
SH
Enz
Tetrahedral intermediate
CoA SH
Dihydrolipoyl
transacetylase
CH3
N
S
CH3
CoA-S
H
R1
R2
C
Enz
C O
B:
S
BH+
SH
TPP
Enz
Acetyl-dihyrolipoamide (Thioester)
100
Oxidation step
HS
SH
Enz
Reduced (dihyrolipoamide)
FAD
CH3
C
NADH + H+
Dihydrolipoyl DH
O
SCoA
Acetyl-CoA
FADH2
S
NAD+
S
Enz
Oxidized (dihydrolipoamide(
101
Mechanism of PDH complex, Cont’d
• This is followed by nucleophilic attack by CoA
on the carbonyl-carbon
• The result is transfer of the acetyl group from
lipoamide to CoA
• The subsequent oxidation of lipoamide is
catalyzed by the FAD–dependent
dihydrolipoyl dehydrogenase and NAD+ is
reduced
102
Regulation of the PDH
Complex
103
Regulation of the PDH Complex
• The reactions of the PDH complex serves to
interconnect the metabolic pathways of
glycolysis, GNG and fatty acid synthesis to
the TCA cycle
• As a consequence, the activity of the PDH
complex is highly regulated by a variety of
allosteric effectors and by covalent
modification
104
Regulation of the PDH Complex,
Cont’d
• The importance of the PDH complex to the
maintenance of homeostasis is evident from
the fact that although diseases associated
with deficiencies of the PDH complex have
been observed, affected individuals often do
not survive to maturity
• Since the energy metabolism of highly
aerobic tissues such as the brain is
dependent on normal conversion of pyruvate
to acetyl-CoA
105
Regulation of the PDH Complex,
Cont’d
• Aerobic tissues are most sensitive to
deficiencies in components of the PDH
complex
• Most genetic diseases associated with PDH
complex deficiency are due to mutations in
PDH
• The main pathologic result of such mutations
is moderate to severe cerebral lactic acidosis
and encephalopathies.
106
Regulation of the PDH Complex
107
Regulation of the PDH Complex,
Cont’d
• NADH and acetyl-CoA, are negative allosteric
effectors on PDH-a, the non-phosphorylated,
active form of PDH
• These effectors reduce the affinity of the
enzyme for pyruvate, thus limiting the flow of
carbon through the PDH complex
• NADH and acetyl-CoA are powerful positive
effectors on PDH kinase, the enzyme that
inactivates PDH by converting it to the
phosphorylated PDH-b form
108
Regulation of the PDH Complex,
Cont’d
• Since NADH and acetyl-CoA accumulate
when the cell energy charge is high, it is not
surprising that high ATP levels also upregulate PDH kinase activity, reinforcing
down-regulation of PDH activity in energy-rich
cells
• Note, however, that pyruvate is a potent
negative effector on PDH kinase, with the
result that when pyruvate levels rise, PDH-a
will be favored even with high levels of NADH
and acetyl-CoA
109
Regulation of the PDH Complex,
Cont’d
• Concentrations of pyruvate which maintain
PDH in the active form (PDH-a) are
sufficiently high so that, in energy-rich cells,
the allosterically down-regulated, high Km
form of PDH is nonetheless capable of
converting pyruvate to acetyl-CoA
• With large amounts of pyruvate in cells
having high energy charge and high NADH,
pyruvate carbon will be directed to the 2 main
storage forms of carbon (glycogen via GNG
and fat production via fatty acid synthesis)
110
Regulation of the PDH Complex,
Cont’d
• Although the regulation of PDH-b
phosphatase is not well understood, it is quite
likely regulated to maximize pyruvate
oxidation under energy-poor conditions and
to minimize PDH activity under energy-rich
conditions
111
Structure of Dihydrolipoly
Transacelyase
• Domain structure of the
dihydrolipoyl
transacetylase (E2)
subunit of the PDH
complex
112
Structure of Dihydrolipoly
Transacelyase, Cont’d
• X-Ray structure of a
trimer of A. vinelandii
dihydrolipoyl
transacetylase (E2)
catalytic domains
113
Structure of Branched-chain aKeto Acid DH Complex
• X-Ray structure of E1
(PDH) from P. putida
branched-chain a-keto
acid dehydrogenase
• The a2b2
heterotetrameric protein
• The TPP binds at the
interface between a and
b subunits
114
Structure of Branched-chain a-Keto
Acid DH Complex, Cont’d
• X-Ray structure of E1
(PDH) from P. putida
branched-chain a-keto
acid dehydrogenase
• A surface diagram of the
active site region
• The lipoyl-lysyl armof the
E2 lipoyl domain has
been model into channel
• The TPP-substrate
adduct in an enamineTPP form
115
Structure of Dihdrolipoamide DH
• X-Ray structure of
dihydrolipoamide
dehydrogenase (E3) from P.
putida in complex with FAD
and NAD+
• The homodimeric enzyme
• One subunit is gray and the
other is colored according to
the domain with its FADbinding domain
116
Structure of Dihdrolipoamide DH,
Cont’d
• X-Ray structure of
dihydrolipoamide
dehydrogenase (E3) from
P. putida in complex with
FAD and NAD+
• The active site of the
enzyme region
• The redox-active portions
of the bound NAD+ and
FAD is shown
117
Mechanism of Dihydrolipoyl DH
• Catalytic reaction cycle
of dihydrolipoyl
dehydrogenase
• It is similar to the
catalytic reaction cycle
of glutathione reductase
• However, glutathione
reductase uses NADPH
instead of NAD+
118
Catabolism of Branched-Chain
Amino Acid
O
O
SCoA
O
O
O
NH3
O
Isoleucine
a-Ketoacid DH
Complex
O
O
SCoA
O
O
O
NH3
CoA
CO2
O
Leucine
O
O
NH3
Valine
SCoA
O
O
O
O
119
Transketolase
120
• The pentose phosphate pathway (PPP)
harvests energy from fuel molecules and
stores it in the form of NADPH
• NADPH is an important electron donor in
reductive biosynthesis
• The PPP also produces 5-carbon sugars
such as ribose which is used in the synthesis
of DNA and RNA
121
• TPP is the coenzyme for the enzyme
transketolase
• Transketolase transfers a 2-carbon unit from
an a-ketose (a sugar with a carbonyl group at
position 2) to an aldose
• 2-carbon unit from the 5-carbon a-ketose
xylulose 5-phosphate is transferred to the 4carbon aldose erythrose 4-phosphate to
make the 6 carbon a-ketose fructose 6phosphate
122
• Glyceraldehyde 3-phosphate results from the
3-carbon fragment that is cleaved from
xylulose 5-phosphate
• Carbanion at C-2 of TPP is first produced
• This carbanion attacks the carbonyl carbon of
the a-ketose to give an addition product
• After deprotonation of the appropriate
hydroxyl group an aldose (in this case
glyceraldehyde 3-phosphate) is released and
an activated glycoaldehyde bound to TPP is
produced
123
• The thiazole nitrogen serves as electron
sink in the reaction and contributes to
resonance stabilization of the resulting
product (activated glycoadehyde)
• Following another deprotonation the nascent
a-ketose is released from TPP
124
Reaction of Transketolase
CH2OH
H
CH2OH
C
O
C
H
C
OH
transketolase
HO
H
C
C
H
OH
CH2-OPO 3H2
D-xylulose-5-phosphate
C
O
HO
C
H
H
C
OH
H
C
OH
CH2-OPO 3H2
H
C
OH
3-phosphoglyceraldehyde
O
H
C
OH
H
C
OH
TPP
CH2-OPO 3H2
D-ribose-5-phosphate
H
O
C
+
H
CH2-OPO 3H2
septulose-7-phosphate
125
C
OH
Structure of Transketolase
3- D Structure of yeast
126
Structure of Transketolase
• Baker's yeast
(Saccharomyces
cerevisiae)
• The coloring scheme
highlights the 2nd structure
and reveals that
transketolase is a dimer
• TPP has been substituted
by 2,3'-deazo-thiamin
diphosphate which is
shown
• Ca2+ (blue-gray) can be
seen complexed with the
diphosphates
127
• Transketolase is a homodimeric
enzyme containing two molecules of
noncovalently bound thiamine
pyrophosphate
128
Mechanism of Transketolase
129
Mechanism of Transketolase
CH3
B:
R1
N
S
R2
R1
CH3
C
Enz
N
H
S
1
R2
C
CH2OH
C
O
HO
C
H
H
C
OH
BH
CH2O P
130
Xylulose-5-phosphate
R1
CH3
CH2OH
N
S
R2
H
B:
C
C
OH
O
C
H
H
C
OH
R1
CH3
N
..
S
CH2OH
C
OH
C
R2
CH2O P
Ribose-5-phosphate
O
H
C
R1
CH3
H
CH2OH
C
OH
N
Dihydroxyethyl-TPP
S
C
C
OH
Glyceraldehyde3-phosphate
R2
O
H
C
BH
H
Ribose-5-phosphate
CH2O P
C
OH
3
CH2O P
131
CH3
R1
CH2OH
N
C
C
O
HO
C
H
H
C
OH
S
R2
H
B:
3
CH2O P
CH3
R1
N
S
CH2OH
C
O
O
C
H
H
C
OH
Sedoheptulose-7-phosphate
R2
3
CH2O P
C
Carbanion-TPP
132
Coenzyme A
133
Vitamin B5 (Pantothenic Acid)
• Pantothenic acid is also known as vitamin B5
• Pantothenic acid is formed from balanine
and pantoic acid
• Pantothenate is required for synthesis of
CoASH
134
Biosynthesis of CoASH
135
136
137
Biosynthesis of CoASH, Cont’d
• Coenzyme A (CoASH or CoA) is synthesized
in a five-step process from pantothenate:
(1) Pantothenate is phosphorylated to 4'phosphopantothenate by the enzyme
pantothenate kinase
(2) A cysteine is added to 4'-phosphopantothenate
by the enzyme phosphopantothenoylcysteine
synthetase to form 4'-phospho-Npantothenoylcysteine (PPC)
138
Biosynthesis of CoASH, Cont’d
(3) PPC is decarboxylated to 4'-phosphopantetheine
by phosphopantothenoylcysteine decarboxylase
(4) 4'-phosphopantetheine is adenylylated to form
dephospho-CoA by the enzyme
phosphopantetheine adenylyl transerase
(5) Finally, dephospho-CoA is phosphorylated using
ATP to coenzyme A by the enzyme dephosphocoA kinase
139
Function of CoASH
• Since coA is chemically a thiol, it can react
with carboxylic acids to form thioesters, thus
functioning as an acyl group carrier
• It assists in transferring fatty acids from the
cytoplasm to mitochondria
• A molecule of coA carrying an acetyl group is
also referred to as acetyl-CoA
• When it is not attached to an acyl group it is
usually referred to as 'CoASH' or 'HSCoA'
140
Function of CoASH, Cont’d
• CoA itself is a complex and highly polar
molecule, consisting of adenosine 3',5'diphosphate linked to 4-phosphopantethenic
acid (vitamin B5( and thence to βmercaptoethylamine, which is directly
involved in acyl transfer reactions
• The adenosine 3’,5’-diphosphate moiety
functions as a recognition site, increasing the
affinity of CoA binding to enzymes
141
Function of CoASH, Cont’d
• Not only is CoA associated intimately with
most reactions of fatty acids, but it is also a
key molecule in the catabolism of
carbohydrates via the TCA cycle in which
acetyl-CoA is a major end-product
• The genes encoding the enzymes for coA
biosynthesis have been identified and the
structures of many proteins in the pathway
have been determined
142
Function of CoASH, Cont’d
• Although there are substantial sequence
differences between prokaryotes and
eukaryotes, coA is assembled in five steps
from pantothenic acid in essentially the same
way in both groups
• However, pantothenic acid per se can only be
synthesized by microorganisms and plants
and must be acquired from the diet by
animals
143
Acyl Carrier Protein (ACCP)
• 4-Phosphopantetheine moiety, linked via its
phosphate group to the hydroxyl group of
serine, is the active component in another
important molecule in lipid metabolism, acyl
carrier protein
• This is a small protein (8.8 kDa), which is part
of the mechanism of fatty acid synthesis
• However, the final step in fatty acid synthesis
in may types of organism is transfer of the
fatty acyl group from ACP to CoA
144
Structure of CoASH
145
Structure of CoASH
146
Deficiency of Pantothenic
• Deficiency of pantothenic acid is extremely
rare due to its widespread distribution in
whole grain cereals, legumes and meat
• Symptoms of pantothenate deficiency are
difficult to assess since they are subtle and
resemble those of other B vitamin
deficiencies
147
Biochemical Features of Coenzyme A
• Nature's ester enolate equivalent is CoA
• It carries a free thiol group to which carboxylic
acid residues are transferred
• Thioesters are very reactive intermediates
• They are both activated towards nucleophilic
attack (electrophilicity of the carbonyl group)
and abstraction of a proton (acidity of the aproton)
148
Biochemical Features of Coenzyme A
Good leaving
group
Acyl transfer reaction
Enolization reaction
149
Biochemical Features of Coenzyme A
• The Claisen reaction between acetyl-CoA and
malonyl-CoA illustrates how b-keto esters are
built up by nature using the enolate derived
from acetyl-CoA as nucleophile
150
Biochemical Features of Coenzyme A
151
Activation of Carboxylate Anion
• How do living systems synthesize the amide
bonds found in proteins, or the ester
functional groups found in lipids, oils and
other natural products?
• Carboxylic acid is not suitable for transferring
acyl goup at physiological conditions and
should be activated
• Many reactions in metabolism involve acylgroup transfer or enolization of carboxylic
acid that exit as unactivated carboxylate
anion at the physiological pH
152
Activation of Carboxylate Anion
• The general mechanism is to make an
activated acyl derivative containing a good
leaving group, and then to carry out an acyl
transfer reaction
• Activation and transfer of acyl groups is a
common mechanism found in proteins, fatty
acis biosynthesis and polyketide natural
products biosynthesis
153
Activation of Carboxylate Anion,
Cont’d
Good leaving
group
O
R
X
Activation
C
O
Carboxylic acid
R
C
Acy
transfer
Y
R
O Acceptor Y
C
O
Activated
carboxylic group
154
Activation of Carboxylate Anion,
Cont’d
• The predominant mechanism by which
carboxylic acids are activated for acyl transfer
and enolization is esterification with the thiol
group of CoASH
• CoASH is well suited to carry out acyl
reactions, since thiols are inherently more
nucleophilc than alcohols or amines
• Thiols are also better leaving groups (pKa 89), which explains why the hydrolysis of
thioesters under basic conditions is more
rapid that oxyester hydrolysis
155
Activation of Carboxylate Anion by
CoASH
Good leaving
group
OH
R
C
BH
B:
SCoA
R
OH
SCoA
+
H
O
B:
R
C
O
Tetrahedral
intermediate
H2O
C
SCoA
O
Thioester
(Acyl-CoA)
156
Acyl Transfer Using Acyl-CoA
Y = alcohol,
amine, carbanion,
or thiol group
Good leaving
group
B:
R
C
SCoA + R` Y
H
O
Thioester
BH
R` Y
R
C
SCoA
O
Acceptor for
acyl group
Tetrahedral
intermediate
R` Y
R
C + CoASH
O
Acylester
157
• Nucleophilic attack to the neutral
activated acyl group is a favored
process and CoA is a good leaving
group from tetrahedral intermediate
158
Thioester vs Oxyester
• Why thioesters in preference to oxyesters?
• The enzymatic reaction don’t use oxyesters,
but use a thioester derived from CoA
• It is advantageous to use thioester in
condensation (Claisen) reactions because the
carbonyl carbon atom has more positive
character than the carbonly in the
corresponding oxyester
159
Thioester vs Oxyester, Cont’d
• Consider the resonance forms for an oxyester
bellow:
..
..
O
O
O
O
..
..
..
R
O
R
O R
R C ..
O
R C ..
R C O R
R C +
..
1
I
1
1
1
II
III
• The contribution from form II tends to
decrease the positive charge on the carbon
160
Thioester vs Oxyester, Cont’d
• However, for thioester, the contribution form II
is less important, whereas I and III may be
more important than the oxyester
• The carbonly carbon of the thioester is more
positive than that the oxyester
..
..
O
R
C
..
..S
O
R1
R
C
I
..
..S
O
R1
R
O
C S R1
..
II
R
C
+
..
..S
III
161
R1
Thioester vs Oxyester, Cont’d
• Positive charge on carbon of the thioester will
make it easier for a nucleophilic compound
such as carbanion to attack the carbonyl
group
• It will also make it easier to remove a proton
from the adjacent carbon atom to form a
carbanion
162
Thioester vs Oxyester, Cont’d
Easy to be
deprotonated
H
H
O
C
C
Not easy to be
deprotonated
..
S
..
R
H
Thioester
H
H
O
C
C
..
O
..
R
H
More positive
charge
Oxyester
Less positive
charge
163
• Activation and Coupling
• Formation of an amide from a carboxylic acid and an amine
often results in overall loss of free energy however there is a
high activation energy to be overcome. To make the synthesis at
all viable this energy must be lowered. This is achieved either by
catalysis or by formation of carboxylic acid derivatives (denoted
RCOX), effectively starting at a higher energy on the free energy
reaction pathway (Figure). The nature of the leaving group 'X'
governs the value of the activation energy. It would thus seem
desirable to form amino acid derivatives with a strongly electron
withdrawing 'X', making the carbonyl carbon more prone to
nucleophilic attack and thereby achieving high reaction rates at
ambient temperatures.
164
165
• Initially the azide and chloride groups were proposed
as the 'X' substituent. While the azide proved to be
almost entirely suited to its task, the acid chloride
suffers from the problem of being "over-activated".
Due to the ease of elimination of the chloride ion, the
carbonyl is prone to attack from even weak
nucleophiles. They are thus prone to hydrolysis and
cannot be conveniently stored.
• Another more subtle side reaction is the loss of chiral
integrity at the a-centre of the activated amino acid.
This can occur through two mechanisms:
166
• 1) Direct abstraction of the a-proton.
• 2) Formation of an oxazolone (also known as
an azlactone), increasing the acidity of the aproton.
• Both these situations result in the formation of
a planar carbanion with reprotonation
possible from both faces (Figure).
167
•
•
Carboxyl Protecting Groups.
Protection of the carboxyl group is required for three main reasons:
•
•
1. To enhance the solubility of the peptide in organic solvents.
•
2. To avoid anhydride formation in the presence of activated amino
acids.
•
3. To allow unambiguous activation in the presence of a coupling
reagent (only the unprotected carboxyl group can be activated).
•
168
Pyridoxal Phosphate (PLP)
169
Pyridoxine in Foods
170
Forms of pyridoxal-5`-phosphate
(a) Pyridoxine (vitamin B6), (b) Pyridoxal-5`-phosphate
(PLP), (c) Pyridoxamine-5`-phosphate (PMP) and (d) The
Schiff base that forms between PLP and an enzyme amino group
171
Pyridoxine (vitamin B6)
• The biologically active form of vitamin B6 is
pyridoxal-5-phosphate (PLP), a coenzyme
that exists under physiological conditions in
two tautomeric forms
• PLP participates in the catalysis of a wide
variety of reactions involving amino acids,
including transaminations, a- and bdecarboxylations, b- and g-eliminations,
racemizations, and aldol reactions
172
Pyridoxine (vitamin B6)
• Note that these reactions include cleavage of
any of the bonds to the amino acid a-carbon,
as well as several bonds in the side chain
• The remarkably versatile chemistry of PLP is
due to its ability to:
– (a) Form stable Schiff base (aldimine) adducts
with a-amino groups of amino acids
– (b) Act as an effective electron sink to stabilize
carbanion intermediates
173
Vitamin B6 (Pyridoxine)
• Vitamin (B6), Pyridoxine, 2-methyl-3-hydroxy4,5-bis(hydroxy-methyl)pyridine, is essential
for protein metabolism, and for the formation
of hemoglobin
• Pyridoxine is needed by rats to cure
dermatitis developed on a Vitamin B -free diet
supplemented by thiamine and riboflavin.
• Its absence from diet is also associated with
anemia
• It is needed also by certain bacteria
174
Vitamin B6 (Pyridoxine), Cont’d
• The related compounds:
– Pyridoxamine
– Pyridoxal
• They posse vitamin B6 activity and are much
more active than pyridoxine
• Good sources of Vitamin B6 are rice husks,
maize, wheat germ, yeast and other sources
of vitamin B
• Food contains three natural forms of vitamin
B6: pyridoxine, pyridoxamine, and pyridoxal
175
Vitamin B6 (Pyridoxine), Cont’d
• It exists in different forms; one of those forms,
pyridoxal 5'-phosphate (PLP), serves a
cofactor in many enzyme reactions, including
the transsulfuration pathway, in which
homocysteine is converted to cystathionine
and then to cysteine
176
Vitamin B6 (Pyridoxine), Cont’d
• Pyridoxine is a water-soluble vitamin
• The body metabolizes this to pyridoxal
phosphate (PLP), which the active cofactor
• PLP is involved in many different types of
reactions involving amino acids
• Reactions may involve the a, b, or g carbon of
the amino acid
177
Vitamin B6 (Pyridoxine), Cont’d
•
Reactions at the a-carbon include:
1.
2.
3.
4.
5.
•
Transamination reactions
Aldol cleavages
Deaminations
Decarboxylations
Racemizations
Reactions at the b-carbon include:
6. Eliminations
7. Replacements
178
Vitamin B6 (Pyridoxine), Cont’d
•
Reactions at the g-carbon include:
8. Eliminations
9. Replacements
179
Vitamin B6 (Pyridoxine), Cont’d
• The commercial vitamin form, pyridoxine
hydrochloride, has the hydrochloride added
for stability and increased shelf life. That form
is artificial but is well utilized by most
individuals
• However, the body cannot use pyridoxine
directly
• Two metabolic steps are needed:
180
Vitamin B6 (Pyridoxine), Cont’d
• First, the pyridoxine must be phosphorylated,
that is, phosphate is added to the ringstructure of the molecule
• Pyridoxine, pyridoxal, and pyridoxamine are
all well-absorbed through the mucosa of the
small intestine
• Inside cells, all these forms are
phosphorylated using the enzyme pyridoxal
kinase
181
Vitamin B6 (Pyridoxine), Cont’d
• Magnesium is needed to activate kinase
enzymes-enzymes that phosphorylate
• However, there is published experimental
work, showing in vitro, that this particular
phosphorylating enzyme in human brain
tissue has higher affinity for zinc and higher
activity with zinc than with magnesium
182
Vitamin B6 (Pyridoxine), Cont’d
• After phosphorylation, if the cell started with
pyridoxal, the biochemistry is completed
• The pyridoxal 5-phosphate (PLP) coenzyme
is ready to go to work
• The phosphorylating kinase prefers pyridoxal
over pyridoxine
• The enzyme phosphorylates pyridoxal faster
than it phosphorylates pyridoxine
183
Vitamin B6 (Pyridoxine), Cont’d
• With pyridoxine, the vegetable source form of
B6, this phosphorylation produces pyridoxine
phosphate
• Then next, the pyridoxine phosphate has to
be oxidized by an oxidase enzyme that is
assisted by vitamin B2, riboflavin, as FAD
184
Vitamin B6 (Pyridoxine), Cont’d
• PLP functions as a coenzyme in enzymes
involved in transamination reactions required
for the synthesis and catabolism of the amino
acids as well as in glycogenolysis as a
coenzyme for glycogen phosphorylase
185
Metabolism of Pyridoxine
CH2OH
From
Diet
HO
Pyridoxine
Kinase
O
O
O
P
CH2OH
O
O
HO
N
H
CH3
Pyridoxine
ATP
ADP
N
H
CH3
Pyridoxine phosphate
186
Metabolism of Pyridoxine, Cont’d
O
O
P
O
CH2OH
O
O
Oxidase
O
HO
CHO
P
O
O
HO
N
H
Pyridoxine phosphate
CH3
FAD
FADH2
N
H
CH3
Pyridoxal 5- phosphate
(PLP)
187
Metabolism of Pyridoxine, Cont’d
O
O
P
O
CHO
O
O
Transaminase O
P
CH2NH2
O
O
HO
HO
N
H
Pyridoxal 5- phosphate
(PLP)
N
H
CH3
Asp
OAA
CH3
Pyridoxamine phosphate
(PMP)
188
Metabolism of Pyridoxine, Cont’d
O
O
P
CHO
CHO
O
O
Phosphatase
HO
O
HO
N
H
N
H
CH3
H2O
PLP
Pi
NAD
CH3
Pyridoxal
Dehyrogenase
NADH
COO
HO
O
Excreted in urine
N
H
CH3
4-Pyridoxic acid
189
Tautomeric Forms of Pyridoxal-5Phosphate (PLP)
190
• The seven
classes of
reactions
catalyzed by
pyridoxal-5phosphate
191
Features of PLP
• The Schiff base formed by PLP and acts as
an electron sink to stabilize the carbanion
• All PLP-dependent enzymes, PLP in the
absence of substrate is bound in a Schiff
base linkage with the NH2 group of an
active site of Lys (Internal Schiff base)
• PLP-dependent enzymes, substrate provides
the amine group whereas the enzyme
provides the carbonly group
192
Features of PLP, Cont’d
• One key to PLP chemistry is the protonation
of the Schiff base, which is stabilized by H
bonding to the ring oxygen, increasing the
acidity of the Ca proton (as shown in Rxn 3)
• The carbanion formed by loss of the Ca
proton is stabilized by electron delocalization
into the pyridinium ring, with the positively
charged ring nitrogen acting as an electron
sink
193
Features of PLP, Cont’d
• Another important intermediate is formed by
protonation of the aldehyde carbon of PLP
(as shown in Rxn 5)
• This produces a new substrate-PLP Schiff
base, which plays a role in transamination
reactions and increases the acidity of the
proton at Cb, a feature important in g
elimination reactions
194
Features of PLP, Cont’d
• The stereochimistry of the amino acid formed
is determined by the direction from which the
H+ is added to the quinonoid intermediate
(VIII) which determined
• Rearrangement to a Schiff base with the
arriving substrate is a transaldiminization
reaction
195
Enz
Enz
Enz
Protonation of
Schiff base
Protonation of
aldehyde
carbon
Pyridoxal-5-phosphate (PLP)
forms stable Schiff base
adducts with amino acids and
acts as an effective electron
sink to stabilize a variety of
196
reaction intermediates