Download Mechanism of Succinyl

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
Coenzyme A (CoA or HS-CoA)
3
Biosynthesis of CoA
4
Coenzyme A (CoA or HS-CoA)
• How do living systems synthesize the amide
bonds found in proteins, or the ester
functional groups found in lipids and other
natural products?
• The general strategy is to make an activated
acyl derivative containing a good leaving
group, and then to carry out an acyl transfer
reaction
5
Mechanism of Activation of Acyl
Group and Transfer
Good leaving group
OH
R
X
Activation
R
C
O
Y
Acyl transfer
R
C
O
Acceptor Y
C
O
6
Acyl Carrier Protein
Thiol group is the point of attachment to the acyl group
being transferred, forming a thioester linkage
7
Acyl Transfer Reaction in Protein
Biosynthesis
• The aminoacyl group of amino acids is
activated and transferred during the assembly
of the polypeptide chains of proteins by
ribosomes
• Amino acid activation is carried out by ATPdependent aminoacyltransfer RNA (tRNA)
synthetases
8
Acyl Transfer Reaction in Protein
Biosynthesis
• The aminoacyl-tRNA ester is then
bound to the ribosome and the free
amine used to form the next amide
bond in the sequence of the protein
9
Acyl Transfer Reaction in Protein
Biosynthesis
O
C
H3C
O
O
O
Alanyl-tRNA
Synthetase
C
C
H3C
NH3
ATP
PPi
C
O
P
O
NH3
10
Ad
Acyl Transfer Reaction in Protein
Biosynthesis
O
O
C
H3C
C
O
P
O
C
Ad
H3C
O
NH3
tRNA-OH
AMP
C
O
tRNA
..
NH2
O
C
O
C
N
tRNA
R
n-1
peptide
11
Acyl Transfer Reaction in Protein
Biosynthesis
O
O
C
Ribosome
tRNA
C
H3C
NH
O
C
C
N
R
n-1
peptide
12
Structure of Coenzyme A
Thiol group is the point of attachment to the acyl group
being transferred, forming a thioester linkage
13
Structure of Coenzyme A
14
Functions of Coenzyme A
• CoA is well suited to carry out acyl transfer
reactions, since thoils are inherently more
nucleophilic 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 than ester hydrolysis
15
Functions of Coenzyme A
• Derived from the vitamin pantothenate (Vit
B3)
• Participates in acyl-group transfer reactions
with carboxylic acids and fatty acids
• CoA-dependent reactions include oxidation of
fuel molecules and biosynthesis of carboxylic
acids and fatty acids
• Acyl groups are covalently attached to the SH of CoA to form thioesters
16
Thioester vs Oxyester
• The carbonyl carbon atom has more positive
charge that the carbonyl in the oxygen esters
• To explain this, we should consider the
resonance for an oxyester and thioester
• The contribution from form II of oxyester
tends to decrease the positive charge on the
carbon, whereas forms I an II tend to increase
the positive charge comparing with forms of
thioester
17
Thioester vs Oxyester
• Positive charge on carbon will make it easier
for a nucleophilic group, such as a 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
18
Resonance
O
R
C
O
R
C
..
..
..
..O
..
..S
O
R1
R
C
R
1
R
..
II
..
..
C
I
..
..S
O
C O R1
I
O
R1
..
O R
..
O
R
R
O
..
C S R1
II
O R1
+
III
O
R1
C
..
R
C
+
..
..S
III
19
R1
Classification of Mechanism of
CoA
20
1. Head Activation
• This reaction involving attack of nucleophilic
groups at the acyl carbonyl carbon atom with
transfer of the acyl function to the attacking
group and release of CoA
• This mechanism is called head activation
because the end of acyl function nearest to
the CoA becomes attached to the nucleophile
21
Head Activation Mechanism
O
R
C
O
S
CoA
R
C
S
Nu
+
S
..
Nu
22
CoA
Examples for Head Activation
Mechanism
•
•
•
•
Nu = phosphate: succinly-CoA synthetase
Nu = Amine: glucosamine acyl transferase
Nu = Water: acetyl-CoA hydrolase
Nu = Alcohol: glycerophosphate
acetyltransferase
• Nu = Thiol: lipoate transferase
• Nu = Hydride: acyl-CoA reductase
• Nu = Carbanion: b-ketothiolase
23
2. Tail Activation
• This is reaction involving condensation
of the alkyl carbon of the acyl-CoA by
the alkyl carbon by formation of its
carbanion
• It is called tail activation because the
target group is attached to the acyl
function by the end furthest from the
CoA
24
2. Tail Activation
• This is reaction involving condensation
of the alkyl carbon of the acyl-CoA by
the alkyl carbon by formation of its
carbanion
• It is called tail activation because the
target group is attached to the acyl
function by the end furthest from the
CoA
25
Tail Activation Mechanism
O
O
O
C
OH
O
C
CH3CH
C
S
CoA
O
..
CH3CH
C
S
CoA
26
Tail Activation Mechanism
• The carbanion on the a-C of the propionlyCoA attacks the bicarbonate to make
methylmalonyl-CoA
• The facile character of this reaction is
attributed to the increased acidity of the
thioester compared to the oxyester
• Thioester is 100 – 1000 times more acid
which means that it has a much greater
tendency to undergo proton dissociation at
the methylene function immediately adjacent
to the sulfur
27
Tail Activation Mechanism
• Negative charge that is produced by this
dissociation is stabilized by
delocalization over the carbonyl group
and by the polarizability of the sulfur
• Example: Citrate synthetase
28
3. Siamese Twin Reaction
• Two molecules of acyl-CoA react
together
• One acyl-CoA undergoes head
activation and other undergoes tail
activation
29
4. Addition Reaction
• Reactions involving additions to CoA
group
• Example: Enoyl-CoA hydratase
30
5. Acyl Group Interchage Reaction
• Reactions involving acyl group
interchage
• Example: Acetoacetyl-CoA transferase
31
Mechanism of Succinyl-CoA
Synthetase
(Succinyl Thiokinase )
(Head Activation Mechanism)
32
Reaction of Succinyl-CoA
Synthetase
+
DG = - 2.9
kJ/mol
33
Reaction of Succinyl-CoA
Synthetase
• The conversion of succinyl-CoA to succinate
by succinyl CoA synthetase involves use of
the high-energy thioester of succinyl-CoA to
drive synthesis of a high-energy nucleotide
phosphate, by a process known as
substrate-level phosphorylation
• A high energy succinyl-phosphate
intermediate is formed, with the phosphate
subsequently being transferred to GDP
34
Reaction of Succinyl-CoA
Synthetase
• Mitochondrial GTP is used in a transphosphorylation reaction catalyzed by the
mitochondrial enzyme nucleoside diphospho
kinase to phosphorylate ADP, producing ATP
and regenerating GDP for the continued
operation of succinyl CoA synthetase
• Enzyme has two isoforms in mammals, one
which specifically uses ATP for synthesis, and
one which utilizes GTP
35
Reaction of Succinyl-CoA
Synthetase
• Succinyl-CoA is a high energy thioester
compound
• The D G = - 35.5 kJ/mol for the hydrolysis of
succinly-CoA is comparable to that of ATP (30.5 kJ/mol)
• How does the enzyme couple the exergonic
cleavage of succinly-CoA to the endergonic
formation of GTP?
36
Mechanism of Energy Coupling
37
Experimental Evidence for
Mechanism of Energy Coupling
• By using isotopically labeled ADP
• In the absence of succinly-CoA, the enzyme
catalyzes the transfer of the g-phosphoryl
group from ATP to [14C]ADP, producing
[14C]ATP
• This isotope-exchange reaction suggests the
participation of a phosphoryl-enzyme
intermediate that mediates the reaction
sequence
38
Experimental Evidence for
Mechanism of Energy Coupling
39
Experimental Evidence for
Mechanism of Energy Coupling
• The enzyme catalyzes two different steps; the
favorable oxidation and unfavorable
phosphorylation reactions are coupled by the
phosphoryl-enzme intermediate
• The formation led to the isolation of a
kinetically active phosphoryl-enzme in which
the phosphoryl group is covalently bound to
the N3 position of a His residue
40
Structure of Succinyl-CoA
Synthetase
• The enzyme is an
a2b2 heterodimer;
the functional units
is one ab pair
41
Mechanism of Succinyl-CoA Synthetase
(Head Activation Mechanism)
Head activation
SCoA
C
O
O P
OH
Pi
O
His
O
His
(C H2 )2
O
COO
SCoA
OH
H
O
C
O
(CH2 ) 2
COO
N
Succinyl-CoA
O P
N
H
N
N
Tetrahedral
intermediate
It is the displacement of CoA by Pi which generates another high
energy compound, succinly-phosphate (phosphoester)
42
BH
Mechanism of Succinyl-CoA Synthetase
(Head Activation Mechanism)
O
O P
CoASH
O
O
OH
SuccinlylPhosphat
C
O
His
C O P
(CH2 ) 2
COO
His
O
O
N
N
(CH2 ) 2 OH
N
BH
COO
NH
B:
phosphohistidine
His removes the phosphoryl
group with the concomitant
generation of succinate and
phosphohistidine
43
Mechanism of Succinyl-CoA Synthetase
(Head Activation Mechanism)
His
BH
O
O P
O
GDP
GM O P
COO
CH2
OH
O
N
N
phosphohistidine
OH
GDP
CH2
COO
Succinate
44
Mechanism of Succinyl-CoA Synthetase
(Head Activation Mechanism)
His
GTP
H
N
N
45
Mechanism of Citrate
Synthtase
(Tail Activation Mechanism)
46
• Note that the enzyme
catalyst enables the
coupling of two
chemically independent
reactions:
– The aldol condensation
(with D G = zero) to the
very favorable hydrolysis of
the CoA thiol ester bond
which drives the overall
reaction far towards product
– Unfortunately the resulting
citrate is a tertiary alcohol
which cannot be readily
oxidized
Citrate
OAA
Aldol condensation
Hydrolysis of
thioester
47
•
•
•
•
Citrate Synthase is an enzyme that catalyzes the first step in the citric acid
cycle. Oxaloacetate and acetyl-CoA bind to Citrate Synthase, which then
catalyzes the reaction which joins the two compounds together
In eukaryotes, Citrate Synthase is a dimer, meaning that it is a protein which is
composed of two separate amino acid chains which are not covalently bonded
to each other
The conformation for Citrate Synthase changes when oxaloacetate (OAA) binds
to citrate synthase. This conformational change creates the acetyl-CoA binding
site. Once OAA is bound, the binding constant for acetyl-CoA is increased by a
factor of 20. To see OAA, click
After OAA is bound to the enzyme, acetyl-CoA binds. The enzyme then
catalyses the following reactions: enolization of the acetyl-CoA by removing the
methyl group, a Claisen condensation which joins the enolated acetyl-CoA and
the oxaloacetate creating citryl-CoA, then a thioester hydrolysis which creates
citrate and CoA.
The monomer of Citrate Synthase, pictured in the lower frame of the left side of
this screen shows the citrate synthase enzyme bound to the two products citrate (click
48
49
50
51
Mechanism of Citrate Synthtase
(Tail Mechanism)
SCoA
SCoA
C
O
C
O
His
H C H
OAA
H
Acetyl-CoA
N
B:
N
H
His
CH
H carbanion H
N
COO
C
N
O
BH
CH2
H 2O
COO
OAA
H
O
Acetyl-CoA
His
O
H
SCoA
N
C
HO C
B:
N
H
CH2
COO
CH2
COO
CoASH
His
SCoA
O C OH
N
N
H
B:
COO H
CH2
C
COO
HO CH2
COO
Citrate
CH2
HO C
His
H
N
N
BH
COO
CH2
COO
Tertahedral Intermediate
52
Mechanism of Thiolase
(Siamese Twin Mechanism)
SCoA
SCoA
C
H
Acetyl-CoA
O
C
O
His
H C H
Acetyl-CoA
carbanion H
N
SCoA
N
N
H
His
C H2
B:
C
N
O
BH
CH3
His
SCoA
O
H
C
N
N
BH
CH2
O
C
CH3
SCoA
SCoA
Tertahedral Intermediate
O
C
CH2 + CoASH
O
C
CH3
SCoA
Acetoaceyl-CoA
His
N
N
H
B:
53
Mechanism of thiolase
(Acyl Group Interchange Mechanism)
SCoA
O
C
His
CoASH
AcAc-CoA
CoASH
C
N
CoAS
C H2
His
H
N
O
CH3
N
H
B:
N
AcAc-CoA
SCoA
C
O
His
C H2
O
C
H
N
SCoA
CH3
Tertahedral Intermediate
N
BH
SCoA
2 C
O
CH3
Acetyl-CoA
His
N
N
H
B:
54
Thiamine Pyrophosphate (TPP)
• TPP is a derivative of thiamine (Vit B1)
• 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
55
Thiamine (Vitamin B1) and TPP
56
Mechanism of Pyruvate
Dehydrogenase Complex
B:
R1
CH3
CH3
N
C
H
Enz
1
S
R2
R1
CH3
N
S
R2
TPP
C
CH3
C
O
C
O
R1
BH
C
S
R2
TPP
O
Pyruvate
CH3
CH3
N
C
OH
C
O
O
C O
Pyr DH
S
SH
CO2
Enz
Acetyl-lipoic acid
CH3
Dihydrolipoyltransacetylase
S
R2
B:
CH3
CH3
R1
CH3
N
O
S
S
C
OH
HETPP
S
S
BH
Enz
Lipoic Acid
SH
ACetyl-CoA
HS
C
H
C C O
R2
SCoA
CH3
N
CoASH
C
R1
Enz
SH
Dihydrolipoyl DH
Enz
FAD
FADH2
NAD
NADH + H
57
Mechanism of Transketolase
B:
R1
CH3
CH3
N
S
R2
C
H
Enz
1
R1
CH2 OH
N
C
S
TPP
R2
C
O
S
R2
TPP
CH2 OH
N
BH
OH C H
H C
R1
CH3
C
OH
C
OH C H
OH
H C
CH2O P
OH
CH2O P
Xylulose-5 P
O
H
C
H C
CH2 OH
C
OH
C
(H C
OH
CH2O P
O
R1
CH3
H
CH2OH
N
OH )3
S
R2
C
C
OH
HETPP
CH2O P
Sedoheptulose-7 P
O
B:
CH3
R1
N
S
R2
CH2 OH
H
C C O
OH
C
(H C
H
OH )3
BH
H
C
(H C
OH )3
CH2O P
Ribose-5 P
CH2O P
58
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
• PLP is a prosthetic group for enzymes
catalyzing reactions involving amino acid
metabolism (isomerizations, decarboxylations,
side chain eliminations or replacements)
59
B6 Vitamins and pyridoxal phosphate
(PLP)
60
PLP Reactions
HN
O
P
C
COO
CH CH2
O
H
O
O
OH
O
H
..
O
N
CH3
CH2
O
C
COO
+
NH3
+
PLP
Transamination
H
O
CH3
C
HN
COO
+
NH3
O
+
Racem ization
a
P
COO
OH
O
O
PLP
C
CH CH2
O
CH3
N
H
a-b-elimination
H
ab
HN C
COO
c
O
CH CH2
CH2 HO
O
H3N
C
COO
O
+
OH
O
P
HN
H
a
O
O
CH3
N
PLP
P
C
COO
CH CH2
O
O
H
OH
O
..
O
N
H
CH3
H
PLP-Ala Schiff
H
HN
+
O
P
O
decarboxylation
CH2OH
b
c
H
deformylation
HN
CH
O
CO2
C
O
..
N
H
H
HOCH2CHO
CH3
+
+
CH2O
+
O
P
COO
CH
O
OH
C
O
O
OH
..
N
PLP
CH3
H
+
NH3
Glycine
+
PLP
61
Mechanism of Amino Acid
Transaminase-1
Lys-258 Ala
H2O
Lys
:N
O
ENZ
OH
O
P
O
OH
O
N
H
CH3
N
O
P
H
O
CH
O
C H
O
O
NH2
H2
N C
H
H
PLP
CH3
H
COO
PLP-Enz Schiff (Aldimine)
CH3
Ala
B:
: NH
Lys
2
H
HN
O
OH
O
P
O
P
H
HC H
O
PLP-Ala Schiff
OH
O
O
N
H2
CH3
N
CH3
N
NH2
O
COO
CH CH3
O
BH
C
Lys
H
Pyridoxamine (PMP)
O
H3C
C COO
H3C
N
H3
NH
CH
O
OAA
O
P
Lys
COO
C
Pyr
OH
O
..
O
CH3
N
H
Quinonoid
B:
H3C
BH
P
O
H
C
COO
:N
Lys
H 2O
H2
NH
H CH
O
O
O
H3C
OH
O
N
C
:N
H2
HC H
O
H
O
P
O
Lys
COO
NH
CH3
O
OH
O
H
N
H
CH3
H
B:
Ketimine
Ketimine
62
Mechanism of Amino Acid
Transaminase-2
COO
NH2
H CH
O
O
P
O
O
C
:N
Lys
COO
H2
CH2
CH2
O
COO
N
CH3 OAA
BH
H 2O
PMP
O
:N
H2
NH
HCH
O
H
Lys
COO
C
OH
OH
O
P
O
CH3
N
H
PLP-OAA Schiff (Aldimine)
COO
CH2
H
C
N
H3
COO
Lys
NH
CH
O
O
P
B:
OH
O
O
COO
CH2
CH3
N
H2N
H
C
COO
H
Asp
H
N
CH
O
O
P
O
Lys
OH
O
N
CH3
BH
H
PLP-Enz Schiff base
(Aldimine)
63
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 (a prosthetic group) enzymes catalyze:
(1) Carboxyl-group transfer reactions
(2) ATP-dependent carboxylation reactions
64
Enzyme-bound biotin
• Biotin is linked by an amide bond to the e-amino
group of a lysine residue of the enzyme
• The reactive center of biotin is the N-1 (red)
65
Reaction catalyzed by
pyruvate carboxylase
Two step mechanism (next slide)
Step 1: Formation of carboxybiotin-enzyme complex
(requires ATP)
Step 2: Enolate form of pyruvate attacks the carboxyl
group of carboxybiotin forming oxaloacetate
and regenerating biotin
66
Mechanism of PEP Carboxylase
(ATP-Independent Biotin Carboxylase)
O
O
CH2
O +
HO C
C
O
COO
Bicarbonate
P
O
B:
OH
R
S
Biotin-Enz
HN
PEP
N
CH2
C
H
H
O
B:
HN
N
O
O
O C
O P
CH2
HN
N
O
BH
R
S
S
O
O P
Carboxyl-phosphate OH
COO
R
O
O C
O
H
O
C
O
O
H
COO
Pyruvate enolate
Biotin-Enz
O
O
O
O P
Pi
O
O
BH
O
R
S
C
HN
BH
R
S
OH
N
HN
CH2
O
C
O
N
O
C OH
O
C
O
COO
COO
OAA
CH2
Carboxyl-biotin-Enz
Pyruvate enolate
COO
67
Mechanism of Pyr Carboxylase
(ATP-Dependent Biotin Carboxylase)
O
O
B:
Biotin-Enz
O +
HO C
AD O
P
O
Bicarbonate
OH
R
S
Pyruvate
HN
N
H
H
B:
ATP
CH2
O
O
O C
O P
O
C
H
O
O
Carboxyl-phosphate OH
COO
Pyruvate
CH2
C
O
BH
R
S
COO
S
HN
B:
R
N
HN
H
N
O
O
O C
O P
O
H
CH2
O
O
O
Biotin-Enz
C
O
COO
Pyruvate enolate
O
O P
BH
O
R
S
BH
R
S
C
HN
O
O
Pi
OH
N
HN
CH2
O
C
O
N
O
C OH
O
C
O
COO
COO
OAA
CH2
Carboxyl-biotin-Enz
Pyruvate enolate
COO
68
Folic acid
69
Tetrahydrofolate (THF)
• Vitamin folate is found in green leaves, liver, yeast
• The coenzyme THF is a folate derivative where
positions 5,6,7,8 of the pterin ring are reduced
• 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)
70
Tetrahydrofolate is an important cofactor in nitrogen metabolism
Biotin transfers carbon in its most oxidized state - carbon dioxide
SAM (S-adenosylmethionine) can transfer carbon in its most reduced
state  methyl groups (but the methyl group comes from
5-methyl-tetrahydrofolate)
Tetrahydrofolate transfers one-carbon groups in intermediate
oxidation states and sometimes as methyl groups
71
Note the differences
between
folate and tetrahydrofolate
Close up views of how the
different oxidation states
of carbon are carried
by folate
72
Different Forms of Folate
Note: these are positions 5,6,7,9 and 10
73
Pterin, folate and tetrahydrofolate (THF)
74
Formation of tetrahydrofolate (THF) from folate
75
• One-carbon
derivatives of
THF
Continued next slide
76
77
THF, Vitamin B12 and SAM
His
Sources of carbon
(1 - 5)
Epinephrine
2
Gly
Formimino-Glu
CO2 + NH4+
3
4
Glu + NH4+
Glucose
Formaldehyde
Gly
1
Formate
5
Ser
THF
DHF
+
NADP
NADPH
THF-C
dTMP
A dUMP
B12- CH3
Purine precursors
B12
Homocysteine
D
SAH
CH3
Trp
B
Ser
C
Gly
Purines (C2 and C8)
Meth
SAM
Recipients of carbon
(A - D)
Norepinephrine
Epinephrine
Guanidinoacetate
Creatine
Nucleotides
Methyated nucleotides
Phosphatidylethanolamine
Phosphatidylcholine
Acetylserotonin
Melatonin
78
Metabolic reactions involving synthesis, interconversions and utilization of
single carbon adducts of tetrahydrofolate
Fig. 20.17
Note following reactions:
9 methionine synthase
10, 11 = serine and glycine
catabolism
79
12 thymidylate synthase
Mechanism of Thymidate Synthase
BH+
H
N
H2N
N
N
Cys
CH2
N
H
R
Cys
O
H
R
R
H
NH
O
OH
O P
NH
OH
O P
N
H2C
O
S
H
B:
CH2
N
H
Cys
R
N
H2C
N
N
R
CH2
N
O
S
N5, N10-methylene-THF
H
N
R
N
N
H2N
N
H
O HC
2
S
H
H
N
H2N
..
OCH2
N
O
O
O
OCH2
N
O
O
H
H
H
OH
H
H
O
H
H
H
OH
H
H
BH+
N
H2N
Cys
H
N
S
CH2
N
O
OCH2
H
H
NH
N
O
O
H
H
OH
H
R
O
N
H2N
Cys
H
R
N
B:
OH
H
N
H2C O
H
O P
dUMP
B:
H
N
N
S
N
O
BH+
R
S
R
N
H
H
O H
H2C
NH
OH
O P
Cys
H
CH2
OCH2
N
O
O
O
H
H
H
OH
H
H
H
N
H2N
H
H
N
N
CH2
N
O
N
R
R
H
7,8-DHF
+
O
H3C
NH
OH
O P
OCH2
N
O
O
O
H
H
H
OH
H
H
dTMP
80
Mechanism of Thymidate Synthase
Inhibition
BH+
H
N
H2N
N
..
N
Cys
CH2
N
H
S
R
Cys
R
N
O
Cys
R
H
NH
O
OH
F
O P
R
F
H
NH
OH
N
H2C
O
S
H
B:
CH2
N
H
R
N
H2C
N
N
R
CH2
N
O
S
N5, N10-methylene-THF
H
N
N
H2N
N
H
O HC
2
H
H
N
H2N
O P
OCH2
N
O
O
O
OCH2
N
O
O
H
H
H
OH
H
H
O
H
BH+
H
H
OH
H
H
5-F-dUMP
H
N
H2N
Cys
H
N
S
N
CH2
N
O
F
N
H2C O
B:
H
NH
OH
O P
R
R
OCH2
N
O
O
O
H
H
H
OH
H
H
81
Mechanism of Inhibition of
Biosynthesis of dTMP
O
H3C
H3N
C
H
H
H
N
H2N
COO
N
R
N
N
H
CH2OH
O
CH2NH
N
H
O P
OCH2
+
N
O
O
O
CH2NH
O
H
NH
OH
N
R
N
NADP
Serine
N
HN
DHF Reductase 2
H
H
NADPH
THF
H
OH
Thymidylate synthetase
5-F-dUMP
H
dTMP
H
Methotrexate
O
H
H3N
C
H
N
H2N
COO
N
N
H
H2
O HC
2
Glycine
NH
OH
N
CH2
N
O P OCH2
R
N
O
O
O
R
H
N5, N10-methylene-THF
H
H
OH
H
H
dUMP
O
F
NH
OH
O P
OCH2
N
O
N
O
COO
O
H
H
OH
H
H
H
5-Fluoro-2'-deoxyuridylate monophosphate (5-F-dUMP)
OOCCH2CH2CHNHC
O
NCH2
N
CH3
N
NH2
N
NH2
Methotrexate
82
Relationship between THF, Vit B12 and
SAM
ATP
Methionine
3P
THF-CH3
THF
B12
S-adenosylmethionine ( SAM )
Precursor
CH3 - product
B12 -CH3
S-adenosylhomocysteine ( SAH )
Homocysteine
Adenosine
83
Mechanism of Serine
Hydroxymethyltransferase-1
Lys
NH
O
H
H
B:
OH
O
P
O
N
H
N C
H
CH
O
HO H2C
CH3
C COO
NH
H
COO
Lys
:HN2
CH
O
O
CH2
BH
P
OH
O
O
OH
Ser
CH3
N
H
PLP-Sub Schiff (Aldimine)
BH OH
H 2C
C COO
N
H3
NH
CH
O
O
P
Lys
OH
O
..
O
N
CH3
H
Next Page
Quinonoid
THF
H2O
H
N
H2N
N
N
O
CH2NH
C H2
C COO
P
O
O
..
N
N
H2N
CH3
H
PLP-THF Schiff (Aldimine)
N
C COO
R
N
N
THF
O
CH2NH
H
O
P
O
N
H3
NH
CH
O
B:
OH
C
H
H
HC
O
O
Lys
N
H3
BH
NH
H
H
R
N
H
OH
O
N
CH3
H
Ketimine
84
Mechanism of Serine
Hydroxymethyltransferase-2
H
N
H2N
N
R
N
N
H
O
CH2NH
B:
HC H
H C COO
P
P
H
O
N
N
H2N
OH
O
H
OH
..
N
CH3
N
N
CH2
C
N
H2
R
O
5
O
H
N
CH3
H
O
Lys
N
H3
CH
O
HC
O
C COO
NH
NH
O
H
BH
Lys
N
H3
Quinonoid
H2O
N, 10N-methylene-THF
Lys
O
P
O
H C H BH
C H
O
O
COO
N
H3
OH
O
CH
O
N
Lys
N
H3
NH
CH3
O
H
P
O
PLP
OH
O
N
H
O
H
H
CH3
B:
COO
H C H
NH2
Gly
85
Folic acid deficincies
Early 1990s: Epidemiological studies demonstrated correlations between folate
deficiencies and increased risk of myocardial infarctions heart attacks
These same individuals also had elevated levels of homocysteine
Working hypothesis:
homocysteine accumulates in folate deficient individuals because of a decrease in the
ability of the methionine synthase reaction to function (due to lack of THF)
Homocysteine causes heart damage by an unknown mechanism
Folate deficiencies during
embryogenesis cause a
significant proportion of
neural tube defects and
consequent failure of the
nervous system to develop
properly
This is most likely due to
inability to synthesize
adequate amounts of thymine
nucleotides
86
HOMOCYSTINURIA AND HEART DISEASE
some patients with arteriosclerosis have elevated homocysteine
low plasma B vitamins and low dietary intake show increased risk for heart
disease
folate/B12 deficiency common in high risk populations (elderly, smokers)
B vitamins decrease plasma homocysteine
homocysteine may oxidatively damage lipoproteins and endothelia of vessel
walls
controversy remains
87
3
S-Adenosylmethionine Homocysteine-SH methyl-B12
THF
4
CYTOPLASM
Methionine synthase
ATP
Methionine-SCH3
↑ in methyl trap
with B12 deficiency
N5-methyl THF
1
Hydroxy B12
X
From diet
2 Adenosyl B12
Succinyl CoA
Methylmalonyl CoA
mutase
Methylmalonyl CoA increase
Propionyl CoA carboxylase
TCA
cycle
biotin
MITOCHONDRION
Propionyl CoA
Figure 5. Metabolism of cobalamin and associated disorders
(homocystinuria and methylmalonic aciduria)
88
Homocysteine
Homocysteinuria
• Rare; deficiency of cystathionine b-synthase
• Dislocated optical lenses
• Mental retardation
• Osteoporosis
• Cardiovascular disease
death
High blood levels of homocysteine associated with
cardiovascular disease
• May be related to dietary folate deficiency
• Folate enhances conversion of
homocysteine to methionine
89
Dihydrofolate reductase converts folate to tetrahydrofolate by
two successive reductions
Dihydrofolate reductase is the target of a
number of clinically important drugs called
antimetabolites: synthetic compounds that are
structural analogs of a normal metabolite
sulfa drugs in bacterial infections
Anticancer agents
90
Aminopterin
Tetrahydrofolate in the metaboblism
of one-carbon units
Single carbon groups
can be carried on N-5,
N-10, or bridged between
N-5 and N-10
methyl
Carbon units are
obtained from a
variety of sources
BUT most activated
single carbon units
are obtained from the
beta carbon of serine
Once a single carbon unit has been
activated by attachment to
tetrahydrofolate it can be used
directly in a biosynthetic reaction or
it can undergo interconversions to
different oxidation states
methylene
formyl
91
Cobalamin (Vitamin B12)
• Coenzymes: methylcobalamin, adenosylcobalamin
• Cobalamin contains a corrin ring system and a cobalt
(it is synthesized by only a few microorganisms)
• Humans obtain cobalamin from foods of animal origin
(deficiency leads to pernicious anemia)
• Coenzymes participate in enzyme-catalyzed
molecular rearrangements in which an H atom and a
second group on the substrate exchange places
92
Cobalamin (Vit B12) and its coenzymes
(a) Cobalamin.
Corrin ring
(black)
93
(b) Abbreviated
structure of cobalamin
coenzymes
94
Intramolecular rearrangements catalyzed by
adenosylcobalamin enzymes
(a) Rearrangement of an H and substituent X on an
adjacent carbon
95
(b) Rearrangement of methylmalonyl CoA
96
Methylcobalamin participates in the
transfer of methyl groups
97
Methyl-Vitamin B12
OH
OH
CH2
O
Amenine
Con+
DMB
5'-Adenosyl-B12
(5'-Adenosylcobalamine)
CH3
Con+
DMB
Methyl-B12
(methylcobalamin)
CH3
Con+
DMB
Methyl B12 as originally isolated
(cyanocobalamin)
n = 1, 2 or 3
98
Mechanism of Methymalonyl-CoA
Mutase
H
1.
2.
Homolytic cleavage of the CCo2+ bond
Ado
H
.
Co2+
Abstraction of hydrogen atom
from methylmalonyl-CoA
COO
CH
H
C
Ado
O
S
H
C
+
H
C
H
CoA
+
H
C
H
+
.
Co2+
COO
.
CH
C
H
Methyl-malonyl-S-CoA
H
C
O
S
CoA
.
2
DMB
Rearrangement
DMB
3
1
Ado
H
Co2+
.
H
C
Ado
+
H
C
H
+
H
H
COO
C
CH
H
3.
Carbon skeleton
rearrangement
CoA
5'-Adenosyl-B12
(5'-Adenosylcobalamine)
Abstraction of hydrogen atom
from 5`-deoxyadenosine
.
Ado
+
H
H
C
+
H
H
H
COO
C
.
CH
C
O
S
CoA
DMB
Hypothetical intermediates
5
4
Release of succinly-CoA and
reformation of Vit B12
S
Cyclopropyloxy radical
H
5.
O
DMB
Co2+
4.
.
C
DMB
Co3+
H
C
C
S
COO
CH
O H
CoA
Co2+
Succinyl-CoA
.
Ado
+
H
.
C
H
+
H
H
COO
C
CH
C
O H
S
CoA
DMB
99
Lipoamide
• Coenzyme lipoamide is the protein-bound form of
lipoic acid
• Animals can synthesize lipoic acid, it is not a vitamin
• Lipoic acid is an 8-carbon carboxylic acid with
sulfhydryl groups on C-6 and C-8
• Lipoamide functions as a “swinging arm” that carries
acyl groups between active sites in multienzyme
complexes
100
Lipoamide
• Lipoic acid is bound via an amide linkage to the
e-amino group of an enzyme lysine
• Reactive center of the coenzyme shown in red
101
Transfer of an acyl group between active sites
• Acetyl groups attached to the C-8 of lipoamide
can be transferred to acceptor molecules
• In the pyruvate dehydrogenase reaction the
acetyl group is transferred to coenzyme A to
form acetylSCoA
102
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
103
Vitamin A (Retinol)
• Vit A is obtained from liver, egg yolks, milk
products or b-carotene from yellow vegetables
• Vit A exists in 3 forms: alcohol (retinol), aldehyde
and retinoic acid
• Retinol and retinoic acid have roles as protein
receptors
• Rentinal (aldehyde) is a light-sensitive compound
with a role in vision
104
Formation of vitamin A
from b-carotene
105
Vitamin D
• A group of related lipids involved in control of
Ca2+ utilization in humans
• Vitamin D3 and 1,25-dihydroxycholecalciferol
106
Vitamin E (a-tocopherol)
• A reducing reagent that scavenges oxygen and
free radicals
• May prevent damage to fatty acids in membranes
Fig 7.29 Vitamin E (a-tocopherol)
107
Vitamin K (phylloquinone)
• Required for synthesis of blood coagulation proteins
• A coenzyme for mammalian carboxylases that
convert glutamate to g-carboxyglutamate residues
• Calcium binds to the g-carboxyGlu residues of these
coagulation proteins which adhere to platelet surfaces
• Vitamin K analogs (used as competitive inhibitors to
prevent regeneration of dihydrovitamin K) are given to
individuals who suffer excessive blood clotting
108
(a) Structure of vitamin K
(b) Vit K-dependent carboxylation
109
Ubiquinone (Coenzyme Q)
• Found in respiring organisms and
photosynthetic bacteria
• Transports electrons between membraneembedded complexes
• Plastoquinone (ubiquinone analog) functions in
photosynthetic electron transport
110
(a) Ubiquinone,
(b) Plastoquinone
• Hydrophobic tail of each is composed of 6 to 10
five-carbon isoprenoid units
• The isoprenoid chain allows these quinones to
dissolve in lipid membranes
111
• Three oxidation states of
ubiquinone
• Ubiquinone is reduced in
two one-electron steps
via a semiquinone free
radical intermediate.
Reactive center is shown
in red.
112
Protein Coenzymes
• Protein coenzymes (group-transfer proteins) contain
a functional group as part of a protein or as a
prosthetic group
• Participate in:
(1) Group-transfer reactions
(2) Oxidation-reduction reactions where transferred
group is a hydrogen or an electron
• Metal ions, iron-sulfur clusters and heme groups are
commonly found in these proteins
113
Stereo view of oxidized thioredoxin
• Cystine group is on the surface (sulfurs in yellow)
114
Cytochromes
• Heme-containing coenzymes whose Fe(III)
undergoes reversible one-electron reduction
• Cytochromes a,b and c have different visible
absorption spectra and heme prosthetic groups
• Electron transfer potential varies among
different cytochromes due to the different
protein environment of each prosthetic group
115
(a) Heme group of cyt a
116
(b) Heme group of cyt b
117
(c) Heme group of cyt c
118
Absorption spectra of oxidized
and reduced cytochrome c
• Reduced cyt c (blue)
has 3 absorbance
peaks: a,b,g
• Oxidized cyt c (red)
has only a g (Soret)
band
119