Transcript Mechanism of Thymidylate Synthase, Cont`d

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:
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Part 2
Coenzymes-Dependent Enzyme
Mechanism
Professor A. S. Alhomida
1
2
Experimental Evidences for
Stereochemistry Hydride
Transfer is Stereospecific
3
H
CONH2
+
N
R
H D
H C C OH
H D
yeast
AD
H D
CONH2
+
N
R
H O
H C C D
H
50: 50
H H
H C C * OH
H D
100%
H
H D
CONH2
+
N
R
H H
H C C OH
H D
H O
H C C H
H
yeast
AD
CONH2
+
N
R
H H
O
*
H C C O S
H D
O
H D
H C C * OH
H H
HO
stereochemistry
inverted
H
CONH2
N
R
+
H D
H C * C OH
H H
yeast
AD
H H
CONH2
+
H O
H C C D
H
N
R
4
Stereospecificity of ADH
• When the redox transformation involves only
hydrogens, one cannot distinguish the
stereochemical course
• However, if one uses deuterium labeling, one
discovers that:
5
Stereospecificity of ADH
• The pro-R hydrogen of ethanol is removed
• The hydrogen is transferred to the re face of
NAD+ (50 : 50)
• If the reaction is run in reverse, the pro-R H of
NADH is transferred to the re face of
acetaldehyde (100%)
6
Stereospecificity of ADH
• The implication of this stereospecificity is that
when ethanol and NAD+ are bound to the
enzyme, their binding sites must orient them
so that the pro-R H of ethanol is directed
toward the re face of the NAD+
• Therefore, the enzyme must bind at least two
of the groups attached to the prochiral center,
leaving the orientation of the NAD+ ring to
distinguish between the two hydrogens
7
Structure of Active site of ADH
• The active site contains
Zn bound to two Cys
and one His
• The Zn ion binds the
acetaldehyde through
its oxygen atom to
polarize it so that it
more easily accepts a
hydride ion (light blue)
from NADH
8
ADH with bound NAD+
9
ADH with bound NAD+ and
Trifluoroenanol (TFE)
• The zinc (orange) is
coordinated to His-67, Cys174, Ser-48, and a water
molecule (not visible)
• Just above the Zn is the
hydride acceptor ring of
NAD+
• The alcohol binds to the Zn
replacing the water
molecule, and thus must lie
between the Zn and the
pyridinium ring
• Leu-57, Phe-93 (mutated to
Trp in this case), and His-51
form the rest of the binding
pocket
10
HS
HR
O
HR
CONH2
HS
H2N
RO
O
HO
N
O
no free rotation
OH
Syn
RO
HO
N
OH
Anti
Conformation of the nicotinamide cofactor determines if the
pro-R or the pro-S hydrogen is transferred from the cofactor
11
Conformation of NAD+ cofactors
NAD+ from Lactate Dehydrogenase (Pro-R specific)
12
NAD+ from Glyceraldehyde-3-phosphate Dehydrogenase (Pro-S specific)
Stereospecificity at C-4 for some NADDependent DH
Enzyme
Nucleotide
C-4 pro H
Product
ADH
NAD
HR
Acetaldehyde
UDP-glucose
DH
NAD
HS
UDPglucuronate
LDH
NAD
HR
Pyruvate
MDH
NAD
HR
OAA
ICDH(Cyt)
NADP
HR
a-KG
ICDH(Mit)
NAD
HR
a-KG
GAPDH
NAD
HS
1,3-BPG
Glu DH
NADP, NAD
HS
a-KG, NH4+
13
Mechanism of Alcohol
Dehydrogenase
14
Mechanism of Alcohol Dehydrogenase
Cys-46 His-67
Cys-174
Zn2+
Zn2+
The
increases the
acidity of the alcohol, but is
not involved in the redox
reaction
Ser -48
H
O
O
His-61
H
CH3 C
Ethanol
H
N
H
N
H
O
B:
NH2
Electron sink (Stored 2 electrons and one
H+). Source & Where?
+
N
R
NAD+
15
H2O
Alcohol Dehydrogeanse
O
CH3C H
Acetaldehyde
H H
+
..
N
R
NADH
Zn2+
H
H
O
Ser
H
His
O
H
N
N
BH+
16
O
NH2
Glyceraldehyde 3-phosphate
Dehydrogenase
17
Glyceraldehyde 3-phosphate
Dehydrogenase
• GAPDH is one of the key enzymes for glycolysis,
reversibly catalyzes the first glycolytic reaction to
involve oxidation-reduction
• It converts the glyceraldehyde-3-phosphate (G3P)
into the high energy phosphate compound, 1,3
bisphosphoglycerate (BPG), using NAD+ as a
cofactor
• BPG reacts with ADP to from ATP by
phosphoglcerate kinase
18
Glyceraldehyde 3-phosphate
Dehydrogenase
• In addition to its role in glycolysis, GAPDH is
known to be involved in several other nonglycolytic functions as well (e.g. apoptosis,
DNA repair and regulation of histone gene
expression
19
Glyceraldehyde 3-phosphate
Dehydrogenase
• It contains an active site Cys, which helps
explain how the enzyme can be inactivated
with a stoichiometric amounts of
iodoacetamide
• Both glycolytic and non-glycolytic functions
are targeted for drug design
• It is one of the few examples in which the
coupling of an oxidation (favorable Rxs) to
phosphorylation (unfavorable Rxs) process
20
Glyceraldehyde 3-phosphate
Dehydrogenase
• It is a medium-sized tetrameric enzyme
• It resembles several other tetrameric NADdependent oxidoreductases, like LDH, ADH
and MDL; all have characteristic structures in
the NAD-binding region known as "Rossmann
folds", after Michael Rossmann, who first
characterized this class of enzymes
structurally
21
Structure of GAPDH shows the tetramer subunits
22
GAPDH Reaction
DG˚ = 6.3 kj/mol
23
Experimental Evidence for Pi
Role
24
What is the role of Pi?
1. If run with catalytic amount of enzyme and omit
Pi from the mixture of the reaction, no product is
formed
O
H
C
GAPDH
H C OH + NAD+
No product
2
CH2OPO3
3-PG
25
Experimental Evidence for Pi Role,
Cont’d
2. If run with a relatively large amount of
enzyme (so that enzyme-bound
intermediates could be detected) and
omit Pi from the mixture of the reaction,
the results showed at time zero, a rapid
increase in the absorbance was
observed, that is, NADH was formed
3. However, the reaction stopped when all
the enzyme-bound NAD+ has been
reduced
26
Experimental Evidence for Pi Role,
Cont’d
4. At this point, only small amount of the
available free NAD+ and substrate had
been consumed
O
H
C
GAPDH
H C OH + NAD+
2
CH2OPO3
3-PG
OPO32
O
C
H C OH + NADH
2
CH2OPO3
1, 3 bPG
27
Experimental Evidence for Pi Role,
Cont’d
5. When Pi was added, more NADH was
rapidly formed
6. The amount of NADH formed was
equivalent to the amount of Pi added
and considerably larger than the amount
of enzyme present
O
O
H
C
C
H C OH + Pi
2
CH2OPO3
3-PG
OPO32
PAPDH
NAD+
H C OH
2
NADH
CH2OPO3
1, 3 bPG
28
Absorbance (340 nm)
Results of the Experimental
Evidence for Pi Role
2nd burst of NADH formation
Add Pi
1st burst of NADH formation
Time (min)
29
GAPDH Reaction, (Cont’d)
Phosphorylation
step
Oxidation
step
The +ve on the NAD+
may help polarize
the thioester to
facilitate the attack
by Pi
30
Structure of GAPDH shows the active site
includes Cys, His residue adjacent to a bound
31
NAD+
Glyceraldehyde 3-phosphate
Dehydrogenase
• A general base in the enzyme abstracts an H+
from Cys, which attacks the carbonyl C of the
glyceraldehyde, forming a tetrahedral
intermediate
• A hydride leaves from the former carbonyl C
to NAD+ in an oxidation step
• Notice, this is a two electron oxidation
reaction similar to seen in alcohol
dehydrogenase
32
Glyceraldehyde 3-phosphate
Dehydrogenase, Cont’d
• GAPDH reaction is the site of action of
arsenate (AsO43-), an anion analogous to
phosphate
• Arsenate is an effective substrate in this
reaction, forming 1-arseno-3phosphoglycerate (1-Ars-PG), but acyl
arsenates are quite unstable and are rapidly
hydrolyzed
33
Glyceraldehyde 3-phosphate
Dehydrogenase, Cont’d
• 1-Ars-PG breaks
down to yield 3-PG
• The result is that
glycolysis continues
in the presence of
Ars, but the
molecule of ATP is
not made because
this step has been
bypassed
34
Mechanism of Glyceraldehyde
3-phosphate Dehydrogenase
35
Mechanism of Glyceraldehyde 3phosphate Dehydrogenase
H
Cys
O
C
H
SH
His
H C OH
B:
CH2OP
GAP
S
Cys
C O
H
His
H
N
H C OH
N
CH2OP
BH
Tertahedral Intermediate
O
Electron sink (Stored 2 electrons
and one H+). Source & Where?
N
NH2
+
N
R
N
H
B:
Cys increases the
acidity of GAP, but is not
involved in the redox
reaction
NAD+
36
O
O P OH
OH
NADH + H
BH
O
S
Cys
Acyl-thioester
Intermediate
C
H C OH
O
O
CH2OP
H
His
N
N
BH
P OH
OH
37
BH
O
Cys
O
O
S
C
H C OH
H
N
P O
OH
His
CH2OP
N
3-Phosphoglyceryl-Enz Intermediate
1. 3-PG-Enz is a
destabilized acylthioester
2. It undergoes 3phosphoglyceryl transfer
to one of the oxygen of Pi
bound at the active site
38
BH
O
O
O
PO C
O H C OH
CH2OP
1, 3 bPG
Cys
High energy
compound
(Acylphosphate
has DG˚ = 49.3 kj/mol)
SH
His
N
N
BH
39
Mechanism of Energy Coupling
40
Mechanism of Energy Coupling
1. Mechanism of energy coupling is when a
high energy compound is being synthesized
(endergonic reaction), is must be coupled
with to some other (exergonic) reaction by
mean of a common intermediate
2. GAPDH catalyzes two different steps; the
favorable oxidation and unfavorable
phosphorylation reactions are coupled by
the thioester intermediate
41
Mechanism of Energy Coupling,
Cont’d
3. The oxidation reaction is favored by the
deprotonation of the hemithioacetal by His176
4. Thioester intermediate allows a favorable
process to drive an unfavorable reaction
5. Thioester intermediate preserves much of
high energy released in the oxidation
reaction
42
Mechanism of Energy Coupling,
Cont’d
• (A) Hypothetical case
with no coupling
between the two
processes, oxidation
and phosphorlyation.
The 2nd step must
have a large
activation barrier,
making the reaction
very slow
• (B) Actual case with
the two reactions
coupled through a
thioester intermediate
Free Energy Profiles for GAP
Oxidation
43
Riboflavin Coenzymes
44
Riboflavin in Foods
45
Riboflavins
• Flavin adenine dinucleotide (FAD) and flavin
mononucleotide (FMN) are derived from
riboflavin (Vit B2)
• Flavin coenzymes are involved in oxidationreduction reactions for many enzymes
(flavoenzymes or flavoproteins)
• FAD and FMN catalyze one or two electron
transfers
46
Riboflavin and its coenzymes
(a) Riboflavin, (b) FMN (black), FAD (black/blue)
47
Reduction, reoxidation of FMN or FAD
48
Riboflain (Vit B2)
Biosynthesis of FAD
49
Flavin Coenzyme: Vitamin B2, one- and two-electron transfer
NH2
OH
O
OH
OH
HO
OH
N
N
10
1
O
N
N
NH
5
N
4a
O-
OH
O
N
O
N
O
OOH
HO
O
NH
N
O
O
Riboflavin
N
N
Flavin Adenine Diphosphate (FAD)
Flavin Mononucleotide (FMN)
OH
OR
HO
CH3
N
N
OH
NH
N
O
Lumiflavin
O
HO
H
N
N
H H
N
NH2
NH
tricyclic flavin ring system: isoalloxazine
OH
N
N
O
NH
O
5,6,7,8-tetrahydrobiopterin
N
O
P O P O
O-
OH
NH
N
O
HO
P O-
O
HO
5'
O
OH
O
5-Deazaflavins
(more closely related to NAD)
50
N
One and Two Electron Reaction
of Flavin
51
Reaction of Flavin with O2
52
Classifications of Flavoenzymes
1. Oxidases: reduced flavin cofactor re-oxidized directly by O2
2. Dehydrogenase: reduced flavin re-oxidized by another group, i.e.,
FlH-red
R-S-S-R
O2
Flox
H2O2
2 R-SH
R-S-S-R
3. Mixed Function Oxidase: reduced flavin reacts with O2 to give a
flavin-4a-hydroperoxide (Fl-OOH) which oxidizes the substrates
by transferring an oxygen atom to the substrate. Overall, O2 is
“split” with oxygen atom being incorporated in the oxidized
substrate, the other oxygen atom ends up as water
4. Electron-Transfer Flavoproteins (ETF):
53
Oxygen:
1
3
O
O
O
spin: s = 2n +1
spin of an electron = ± 1/2
O
singlet
triplet
For oxidases and mixed function oxidases
R
N
N
H
R
N
N
H
O
N
O
NH
+
O
O
R
N
H+
N
NH
O O
B:
N
O
spin
inversion
O
N
R
N
electrontranfer
3
N
H O O
HO
N
H
O
O
NH
1
+
O
O
O
for
oxidases
R
N
N
+
NH
Flavin-4a-hydroperoxide
H2O2
NH
N
H+
O
O
pKa H2O2 ~ 11.6
For mixed function oxidases, the flavin 4a-hydroperoxide is the
oxygen-transfer (oxidizing) agent
54
For mixed function oxidases (monooxygenases), the oxidized flavin
Reducing agent is often NAD(P)H and is usually supplieds a separate
enzyme
R
N
NAD(P)H
N
O
NH
N
O
NAD(P)
R
N
N
H
N
O
NH
O
R
N
H2NOC
Nicotinamide
E°'~ -0.32 V
H
O
H
N
HN
O
N
R
N
NAD
Flavin
E°'~ -0.20 V
FAD
55
Mechanism of Flaovenzymes
• Three main different mechanisms have been
proposed for the reaction catalyzed by this
flavoenzymes:
• (1) Carbanion formation mechanism: by
abstraction of the H+ of the substrate
• (2) Direct hydride-transfer mechanism
• (3) Concerted mechanism (1 and 2 together)
56
Carbanion Mechanism
57
D-Amino Acid Oxidase
58
D-Amino Acid Oxidase,Cont’d
• D-Amino acid oxidase (EC 1.4.3.3, DAAO)
catalyzes the dehydrogenation of D-isomer of
amino acids to give the corresponding aimino acids and, after subsequent hydrolysis,
a-keto acids and ammonia
• It spreads from yeasts to human and it
is not present in bacteria nor in plants
59
D-Amino Acid Oxidase, Cont’d
• Recently mammalian D-amino acid has been
connected to the brain D-Ser metabolism and
to the regulation of the glutamatergic
neurotransmission
60
Structure of D-Amino Acid
Oxidase
Yeast DAAO
Human DAAO
61
Structure of D-Amino Acid
Oxidase, Cont’d
• D-Ala is located above
the reduced flavin Reside
• (- - -) lines denote
hydrogen bonds
involved in substrate
fixation
Yeast DAAO
62
Stabilization of the Negative Charge in
the Active Site by Arg-285
DAAO + D-Ala
Yeast DAAO
63
Potential of the DAAO-based
Selection system
64
Potential of the DAAO-based Selection
System
65
Potential of DAAO-based Selection
System, Cont’d
• (a) Growth of nontransgenic wild-type plants
lacking DAAO activity is inhibited by D-amino
acids such as D-Ala but is not affected by
others such as D-Ile
• In contrast, plants expressing the transgenic
DAO1 gene detoxify D-Ala and survive
(positive selection), whereas they metabolize
D-Ile to toxic compounds that kill the plants
(negative selection)
66
Potential of DAAO-based Selection
System, Cont’d
• (b) Plants that have integrated a gene of
interest together with the DAO1 marker are
first detected by positive selection
• Subsequent negative selection identifies
plants from which the no-longer-desirable
selection marker has been removed (e.g., by
genetic segregation or site-specific
recombination systems), leaving the gene of
interest as the only transgenic sequence in
place
67
Structure of D- and L-Alanine
L-Alanine
D-Alanine
68
Structure of D- and L-Alanine (Cont’d)
L-Alanine
D-Alanine
69
D-Amino Acid Oxidase
• D-Amino acids are not normal metabolites in
mammalian cells
• D-Amino acids librated from bacterial cells
that have been lysed by macrophages
• D-AA oxidase catalyzes the reaction by Nnucleophile mechanism but not by Cnucleophile mechanism because X-ray
structure shows no active site base
70
D-Amino Acid Oxidase,Cont’d
• The standard reduction potential of the flavin
in D-amino acid oxidase, a flavoprotein, is
about 0.0 V
• Remember, the more positive the standard
reduction potential, the more likely the
substrate will be reduced and hence act as
an oxidizing agent
• FAD in D-AA oxidase is a better oxidizing
agent than free FAD
71
D-Amino Acid Oxidase, Cont’d
• The Kd for binding of FAD to the enzyme is
10-7 M compared to the Kd for binding of
FADH2, which is 10-14 M
• By gaining electrons, the FAD binds more
tightly, which preferentially stabilizes the
bound FADH2 compared to the bound FAD
• This shifts the equilibrium of FAD → FADH2
to the right, making the bound FAD a stronger
oxidizing agent
72
Reaction of D-Amino Acid Oxidase
H
H
OH
CH3
C
C
NH3
O
Enz-FAD
OH
C
C
O
Enz- FAD
Enz- FADH2
CH3
CH3
NH3
Reox
Step
OH
C
C
NH2
O
D-Alanine
73
Reaction of D-AA Oxidase, Cont’d
FAD
Enz
O2
OH
C
C
O
H2O2
CH3
NH2
Enz-FAD
+
H2O2
Hydrogen peroxide
74
Reaction of D-AA Oxidase, Cont’d
OH
CH3
C
NH2
C
O
Pyruvate imine
H2O
Nonenzymatic Rnx
NH4
OH
CH3
C
C
O
O
Pyruvate
75
Mechanism of D-Amino Acid
Oxidase
76
Mechanism of D-Amino Acid Oxidase
C-nucleophile mechanism. Amino acid enolate adds to the
flavin 4a-position. The flavin then acts as a leaving group
R
N
H2N H
R
N
:B
O
R
N
N
H
R
CO2H
N
O
NH
+
O
R
CO2H
H2N
H2O
H2N
R
O
O
NH
N
H
H2N
pKa~ 25
N
NH
N
H+
CO2H
R
N
O
CO2H
O
R
+
NH4
CO2H
X-ray structure shows NO active site base
77
Mechanism of D-Amino Acid
Oxidase
(Hydride Transfer Mechanism)
N-Nucleophile Mechanism
78
Arg-285
C
NH2
H2N
BH+
B:
R
H3C
H3C
N
N
NH
N
OH
O
FAD
B:
Ser-335
O
H
H
H N
C
H
CH3
D-Amino Acid
Redox step
R
H
H3C
N
N
H3C
N
FADH2
COO
O
O2
H
O
NH
O
BH+
O
H
N
C
H
CH3
COO
Pyruvate imine
Electron sink (Stored 2 electrons and 2 H+). Source & Where?
79
Mechanism of D-AA Oxidase, Cont’d
R
H3C
N
H3C
N
N
O
NH
BH+
O
H
B:
OH
O
H2O 2
Hydrogen peroxide
R
H3C
N
H3C
N
N
O
NH
O
FAD
80
Mechanism of D-AA Oxidase, Cont’d
OH
CH3
C
NH2
C
O
Pyruvate imine
H2O
Non-enzymatic Rxn
NH4
OH
CH3
C
C
O
O
Pyruvate
81
p-hydroxybenzoate
Hydroxylase
82
p-hydroxybenzoate Hydroxylase
• p-Hydroxybenzoate hydroxylase (EC
1.14.13.2) is a flavoprotein involved in
degradation of aromatic compounds, and it
has become a model for enzymes involved in
the oxygenation of a substrate
• It is an important enzyme in the microbial
biodegradation of a wide variety of aromatic
chemicals, including pollutants and lignin, a
major component of wood and so among the
most abundant of all biopolymers
83
p-hydroxybenzoate Hydroxylase,
Cont’d
• Enzyme structure is unusual because there is
no recognizable domain for the binding of
NADPH involved in the reaction
• The flavin ring structure moves substantially
in the active site, probably to enable
substrate and product exchange into this site
and possibly to regulate the reduction of the
flavin by NADPH
84
p-hydroxybenzoate Hydroxylase,
Cont’d
• A chain of H-bonds can connect p-hydroxybenzoate in the active site of the enzyme with
the protein surface
• This chain is responsible for the reversible
formation of substrate phenolate anion
observed in the active site and partly
responsible for the reactivity of this substrate
85
Stabilization of Transition State of phydroxybenzoate Hydroxylase
• An OH group (center) is
transferred from a flavin
cofactor (right) to the
substrate (left)
• The transition state is
stabilized by a
hydrogen bond
interaction with a key
group in the enzyme
(shown as a dotted line)
86
Reaction of p-hydroxybenzoate
Hydroxylase
HO
COO + O2
Hydroxylase
Enz-FADH2
4-hydroxybenzoate
COO
H2O + HO
HO
Enz-FAD
3,4-dihydroxybenzoate
Reductase
NADP
NADPH
87
Reaction Steps of phydroxybenzoate Hydroxylase
Reduced form
Reduced form
88
Mechanism of phydroxybenzoate Hydroxylase
(Monooxygenase)
89
Mechanism of p-hydroxybenzoate
Hydroxylase (Monooxygenase)
H3C
H3C
R
H
N
N
R
O
NH
N
H
B:
O
H3C
N
H3C
N
H
FADH2
O2
O
H
N
O
NH
O
BH+
O
Electron sink (Stored 2 electrons and 2 H+). Source & Where?
90
Mechanism of p-HB Hydroxylase (Cont’d)
R
H3C
N
H3C
N
H
N
Electrophilic oxygen
O
NH
O
O
OH
BH+
O
H
CCO
Hydroxybenzoate
R
H3C
N
H3C
N
N
O
NH
BH+
O
H
B:
O
H
O
HO
+
BH
H
B:
CCO
91
Mechanism of p-HB Hydroxylase, Cont’d
NADPH
HO
HO
H2O +
COO
BH+
R
H3C
N
H3C
N
N
O
NH
O
H O
H
..
FAD
NH2
N
Electron sink (Stored 2 electrons
and 2 H+). Source & Where?
R
NADPH
92
Mechanism of p-hydroxybenzoate
Hydroxylase, Cont’d
O
NH2
+N
Reductase
R
NADP+
R
H
H3C
N
N
H3C
N
O
NH
O
H
FADH2
93
Glutathione Reductase
94
Biosynthesis of Glutathione
95
Function of Glutathione
• Glutathione (GSH) is an important tripeptide
(Glu, Cys, and Gly ) present in significant
concentrations in all tissues
• The function of GSH is to protect cells from
oxidative stress or the presence of ROS
which might otherwise damage them
• The oxidizing agents react with the -SH group
of Cys of the GSH instead of doing damage
elsewhere
96
Glutathione Reductase
• Many foreign chemicals get attached to GSH,
which is really acting as a detoxifying agent
• GSH reductase (from human RBC) is dimer
of identical 478-residue subunits (52.4KD per
monomer) are covalently linked by an
intersubunit disulfide bond
• Each subunit contains FAD- and NADPHbinding domains that composed of a babab
97
Glutathione Reductase
• The two S atoms of
the redox-active
residues are in
yellow spheres
• The FAD prosthetic
groups are in
orange color near
the active disulfide
bridge
98
Glutathione Reductase
• Each subunit is
organized into five
domains
• The two subunits
are covalently linked
by a disufide bridge
• The binding sites for
NADPH, GSSG and
FAD are indicated
99
Glutathione Reductase Catalytic Cycle
100
Reaction of Glutathione Reductase
GS-SG
101
Mechanism of Glutathione
Reductase
(Indirect-NAD-Hydride Transfer)
102
Mechanism of Glutathione Reductase
(Indirect-NAD-Hydride Transfer)
Cys-58
Cys-63
S
S
S
His
R
H3C
N
H3C
N
N
O
NH
O
FAD
3H
H
N
N
HO
..
BH+
NADP+
S
R
H
H3C
N
N
H3C
N
FADH2
3H
O
NH
O
N
N
H
B:
NH2
N
R
NADPH
Electron sink (Stored 2 electrons and 2 H+). Source &
103
Where?
Mechanism of Glutathione
Reductase, Cont’d
S
G S
S
H3C
H3C
G
S
R
H
N
N
NH
N
O
GSSG
O
3
H
N
BH+
N
104
Mechanism of Glutathione
Reductase, Cont’d
G S
GS3H
S
S
R
H
H3C
N
N
H3C
N
O
NH
O
N
N
H
B:
105
Mechanism of Glutathione
Reductase, Cont’d
G S
S
S
S
R
H3C
N
H3C
N
S
R
N
O
NH
O
H
H3C
N
N
H3C
N
N
NH
O
N
O
N
+
BH
N
H
GSH
106
NAD/NADH versus FAD/FADH2
107
NAD/NADH vs FAD/FADH2
• FAD binds tightly to the enzymes (sometimes
covalently attached to Cys or His through C8a) so as to control the nature of the
oxidizing/reducing agent that interact with
them
• Because FADH2 is susceptible to reaction
with dioxygen (O2)
• FAD/FADH2 can form stable free radicals
arising from single electron transfers
108
NAD/NADH vs FAD/FADH2, Cont’d
• O2 in the cell won't react with FAD in the
cytoplasm
• If bound FAD is used to oxidize a substrate,
the enzyme would be inactive in any further
catalytic steps unless the bound FADH2 is
reoxidized by another oxidizing agent
109
NAD/NADH vs FAD/FADH2, Cont’d
• FAD has a more positive reduction
potential than NAD
• It is used for more demanding oxidation
reactions, such as dehydrogenation of a
C-C bond to form an alkene (C = C)
110
NAD/NADH vs FAD/FADH2, Cont’d
• FAD can exit as FAD or FADH2 whereas NAD
can exit as NAD or NADH
• FAD can carry out 1 electron and 1 proton or
2 electrons and 2 protons whereas NAD can
carry out only 2 electrons and 1 proton
111
NAD/NADH vs FAD/FADH2, Cont’d
• The standard reduction potential for flavin
enzymes varies from - 465 mV to + 149 mV
• Because the FAD is tightly bound to the
enzyme so its tendency to acquire electrons
depends on its environment
• Comparing to the reduction potential of free
FAD/FADH2, which in aqueous solution is 208 mV
112
NAD/NADH vs FAD/FADH2, Cont’d
• The standard reduction potential of the flavin
in D-amino acid oxidase is about 0.0 V
• FAD in D-amino acid oxidase is a better
oxidizing agent than free FAD
• NADH does not react well with O2, since
single electron transfers to/from NAD/NADH
produce free radical species which can not be
stabilized effectively whereas FAD reacts with
O2
113
Pterin Coenzyme
114
Pterin Coenzyme
• Coenzyme has a 3-carbon side chain at C-6
• Not vitamin-derived, but synthesized by some
organisms
(5,6,7,8, Tetrahydrobiopterin, THB or BH4)
115
Pterin, Folate and Tetrahydrofolate
(THF or FH4)
116
Biosynthesis of Tetrahydrobiopterin
(THB)
117
Biosynthesis of THB, Cont’d
118
Biosynthesis of THB, Cont’d
119
Structure of THB
• THB is the coenzyme for Phe 4-hydroxylase
(PAH), Tyr 3-hydroxylase, andTrp 5hydroxylase; the latter two are key enzymes in
the biosynthesis of biogenic amines
• THB serves as the cofactor for nitric oxide
synthase and glyceryl-ether monooxygenase
• THB can react with O2 to form an active
oxygen intermediate that can hydroxylate
substrates
120
Phenylalanine Hydroxylase
121
Structure of Phenylalanine
Hydroxylase (PAH)
• It catalyzes the conversion of Phe to Tyr
• It is regulated by Phe, THB, and
phosphorylation
• Four subunits of PAH interact to form a
tetramer, which is the functional unit for this
enzyme
• It is non-heme metallenzyme
122
Structure of PAH, Cont’d
• PAH includes a nonheme iron atom at its
active site
• Fe bound to His N, Glu
O and water O
• O2, THB, and the iron
atom in the ferrous
(Fe2) oxidation state
participate in the
hydroxylation
• O2 react initially with
THB to form a peroxy
intermediate
7,8-dihydrobiopterin
Glu
His
His
PDB 1DMW
Phenylalanine
Hydroxylase
123
Structure of PAH, Cont’d
• Tyrosine, an
essential nutrient for
individuals with
PKU, must be
supplied in the diet
Transaminase
Phenylalanine
Phenylpyruvate
(Phenylketone)
Phenylalanine Deficient in
Hydroxylase
Phenylketonuria
Tyrosine
Melanins
Multiple
Reactions
Fumarate + Acetoacetate
124
Structure of PAH, Cont’d
• Each molecule in the tetramer is organized
into three domains:
– Regulatory domain
– Catalytic domain where the enzyme activity
resides
– Tetramerization domain that assembles four
chains into the tetramer
• At the heart of each catalytic domain is an
iron ion (Fe) that plays an important role in
the enzyme action
125
Structure of PAH, Cont’d
126
Structure of PAH, Cont’d
127
Reactions of PAH and THF
128
Deficiency of PAH
• Genetic deficiency of PAH leads to the
disease phenylketonuria (PKU)
• Phe and phenylpyruvate accumulate in blood
and urine
• Mental retardation results unless treatment
begins immediately after birth
• Treatment consists of limiting phenylalanine
intake
129
Deficiency of PAH, Cont’d
• High concentration of Phe:
– Can cause neurologic damage
– Inhibits Tyr Hydroxylase, on the pathway for
synthesis of the pigment melanin from Tyr
– Individuals with PKU have light skin and hair color
• The biosynthesis of the neurotransmitters
(dopamine, adrenaline, and noradrenaline)
from dietary Phe (into Tyr) is initiated by the
PAH
• Tyr becomes an essential nutrient for
individuals with PKU
130
Experimental Evidence for 1,2Shift Mechanism
• An unexpected aspect of the PAH reaction is
that a 3H atom ends up on C3 rather than
being lost to the solvent by replacement for
the OH group
• The mechanism is called NIH shift or 1,2 shift
mechanism
131
Experimental Evidence for 1,2Shift Mechanism (Cont’d)
CO2-
CO2D
NH3
NH3
HO
~50% D- incorporation
D
Electrophilic substitution
analogous to
p-hydroxybenzoate
hydroxylase
CO2-
B:
HO
D
NH3
CO2HO
NH3
This mechanism would
require NO D in the product
132
Experimental Evidence for 1,2Shift Mechanism, Cont’d
OH
H
N
N
NH2
H
OH
N
H
NH
+
NH3
D
NH3
D
CO2-
CO2- 1,2-hydride
shift
CO2O
O O
H
+
D
O
H
HO
B
CO2-
CO2NH3
HO
D H
NH3
HO
H (D)
~50% D- incorporation
133
H
NH3
Mechanism of PAH
(Mono-oxygenase)
134
Mechanism of PAH
(Mono-oxygenase)
H
H2N
N
H
N
H2N
N
B:
N
H
O
H
O O
N
CH CH CH3
N
N
OH OH
N
O
THB
R
H
O
O
Pterin hydroperoxide
Electron sink
(Stored 1 electron
and H+). Source &
Where?
Gl u-330
His-290
H2O
Fe2+
H2O
His-285
H2O
135
Mechanism of PAH, Cont’d
H
H2N
N
N
H2O
H
N
H
H N
N
O
OH
R
BH+
H2O
N
N
N
O
O
Fe2+
N
H
B:
H
R
O
Pterin-4a-carbinolamine
+
H2O
HOH
O
2
Fe4+
Electron sink (Stored 1 electron and 1
H+). Source & Where?
H2O
H2O
Oxyferry
136
Mechanism of PAH, Cont’d
2
O
Fe4+
Fe4+
Phe
H2O
O
H
H2O
H
B:
Oxyferry
H N
N
N
BH+
Electron sink (Stored 1
electron and 1 H+).
Source & Where?
Oxyferry
3
N
Phe
N
O
R
Dehydrogenase
1
R
H
3
2
137
Mechanism of PAH, Cont’d
1
Electron sink (Stored 2
electrons and 1 H+).
Source & Where?
NADPH 3
DHB reductase
NADP+
H
H2N
N
N
N
N
H
O
H
THB
3H
CH CH CH3
OH OH
138
2
H N
H
N
H
B:
N
H
N
N
BH+
R
O
H
H
N
H
N
N
N
N
O
R
DHB
NADPH
Electron sink (Stored 2
electrons and H+). Source &
Where?
DHB reductase
NADP+
H
H2N
N
N
N
N
H
O
H
THB
CH CH CH3
OH OH
139
H
3
O
H
3
R
Epoxide
H
Hydrogen ion migration
H
3
R
H
OH
Carbonion at C3
H
3
H
R
OH
3
H
R
3
OH
H
H
HO
R
Resonance-stabilized oxonium ion
Tyr
140
Tetrahydrofolate (THF)
141
Biosynthesis and Absorption of THF
• Folate is obtained primarily from yeasts and
leafy vegetables as well as animal liver
• Animal cannot synthesize PABA nor attach
glutamate residues to pteroic acid, thus,
requiring folate intake in the diet
• When stored in the liver or ingested folate
exists in a polyglutamate form
142
Folic Acid (Vitamin B9)
We cannot synthesize
143
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)
144
Tetrahydrofolate (THF)
• THF is an important coenzyme in nitrogen
metabolism
• Biotin transfers carbon in its most oxidized
state-carbon dioxide (CO2)
• SAM can transfer carbon in its most reduced
state – methyl groups (but the methyl group
comes from 5-methyl-THF)
• THF transfers one-carbon groups in
intermediate oxidation states and sometimes
as methyl groups
145
THF, Cont’d
• Intestinal mucosal cells remove some of the
glutamate residues through the action of the
lysosomal enzyme, conjugase
• The removal of glutamate residues makes
folate less negatively charged (from the
polyglutamic acids) and therefore more
capable of passing through the basal lamenal
membrane of the epithelial cells of the
intestine and into the blood
146
THF, Cont’d
• Folate is reduced within cells (principally the
liver where it is stored) to THF through the
action of dihydrofolate reductase (DHFR), an
NADPH-depending enzyme
147
Formation of THF from folate
148
Different Forms of THF
• One-carbon
derivatives of
THF
• Note: these
are positions
5,6,7,9 and
10
Continued next slide
149
150
THF, Vitamin B12 and SAM
His
Sources of carbon
(1 - 5)
Epinephrine
2
Gly
Formimino-Glu
3
Glu + NH4
Glucose
CO2 + NH4+
4
+
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 151
Acetylserotonin
Melatonin
THF in the Metabolism 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
152
Folate Deficiencies
• 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
153
Folate Deficiencies, Cont’d
• 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
154
Folate Deficiencies, Cont’d
• 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
155
Homocysteine
156
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
157
Enzyme Deficiencies Causing
Homocystinuria
158
Thymidylate Synthase
159
Reaction of Thymidylate Synthase
160
Thymidylate Synthase
• Thymidylate synthase sits at a junction
connecting dNTP synthesis with folate
metabolism
• It has become a preferred target for inhibitors
designed to inhibit DNA synthesis
• An indirect approach is to employ folate
precursors or analogs as antimetabolites of
dTMP synthesis
• Purine synthesis is affected as well because it
is also dependent on THF
161
Thymidylate Synthase, Cont’d
• Synthesis of dTMP from dUMP is
catalyzed by thymidylate synthase
• The 5-CH3 group is ultimately derived
from the b-carbon of serine
162
Thymidylate Synthase, Cont’d
• 5-Fluorouracil (5-Flu) is a thymine analog
• It is converted in vivo to 5'-fluorouridylate by a
PRPP-dependent phosphoribosyltransferase,
and passes through the reactions of dNTP
synthesis, culminating ultimately as 2'-deoxy5-fluorouridylate, a potent inhibitor of dTMP
synthase
• 5-Flu is used as a chemotherapeutic agent in
the treatment of cancer
163
Thymidylate Synthase, Cont’d
• Similarly, 5-fluorocytosine is used as an
antifungal drug because fungi, unlike
mammals, can convert it to 2'-deoxy-5fluorouridylate
• Further, malarial parasites can use
exogenous orotate to make pyrimidines for
nucleic acid synthesis whereas mammals
cannot
• 5-fluoroorotate is an effective antimalarial
drug because it is selectively toxic to these
parasites
164
Thymidylate Synthase, Cont’d
• Precursors and analogs of folate employed
as antimetabolites:
–
–
–
–
Sulfonamides
Methotrexate
Aminopterin
Trimethoprim
• They bind to DHF reductase with about one
thousand-fold greater affinity than DHF and
thus act as virtually irreversible inhibitors
165
166
167
Thymidylate Synthase, Cont’d
• Sulfa drugs, or
sulfonamides, owe
their antibiotic
properties to their
similarity to paminobenzoate
(PABA), an
important precursor
in folate synthesis
• Sulfonamides block
folate formation by
competing with
PABA
168
Human thymidylate synthase
169
Mechanism of Thymidylate
Synthase
170
Mechanism of Thymidylate Synthase
BH+
H
N
H2N
..N
N
Cys
H
O
S
H
N
H 2C
H
N
H2N
CH2
R
Cys
N
R
O
S
B:
CH2
N
H
N
N
R
N
H 2C
R
H
H
N5, N10-Methylene-THF
O
NH
OH
O P OCH2
N
O
O
O
H
Electron sink (Stored 2 electrons and 2
H+). Source & Where?
H
H
OH
H
H
dUMP
171
Mechanism of Thymidylate
Synthase, Cont’d
BH+
H
N
H2N
H
N
N
CH2
N
H
N
H2C
S
NH
O P OCH2
N
O
H
R
N
O
N
CH2
N
O
S
H
O
OH
R
Cys
O
Cys
N
H2N
H
O H
B:
NH
OH
O P OCH2
N
O
O
O
H
H
H
OH
H
H
H
R
N
H 2C
H
H
OH
H
H
Electron sink (Stored 2 electrons and 2 H+). Source & Where?
172
R
O
Mechanism of Thymidylate
Synthase, Cont’d
Hydrogen transfer
B:
H
N
H2N
Cys
H
N
O
S
N
H
N
CH2
BH+
R
S
R
N
H
Cys
H
NH
O P OCH2
N
H2N
OH
N
O
O
N
N
N
H
O
H
H
OH
H
NH
OH
CH2
N
R
R
O
H
H3C
H
O
H 2C
O
H
H
H
7, 8-DHF
+
O P OCH2
N
O
O
H
H
H
OH
H
dTMP
Electron sink (Stored 2 electrons and 2 H+). Source & Where?
173
H
O
Mechanism of Thymidylate
Synthase Inhibition by 5Fluoro-dUMP
174
Mechanism of Thymidylate Synthase
Inhibition by 5-Fluoro-dUMP
BH+
H
N
H2N
..N
N
Cys
H
O
S
H
N
H 2C
H
N
H2N
CH2
R
Cys
N
N
N
R
O
S
B:
CH2
N
H
R
N
H 2C
R
H
H
N5, N10-Methylene-THF
O
F
NH
OH
O P OCH2
N
O
O
O
H
H
H
OH
H
H
FdUMP
175
Mechanism of Thymidylate
Synthase Inhibition by 5-FluorodUMP
H
N
H2N
H
N
N
H2N
CH2
N
H
N
H2C
S
H
O
F
NH
OH
O P OCH2
N
O
R
H
Cys
O
Cys
N
O
R
N
CH2
N
O
S
F
B:
X
OH
N
O P OCH2
R
N
H 2C O
H
NH
N
O
O
O
O
H
H
H
OH
H
H
H
H
H
OH
H
H
Inhibition by Flu-dUMP results from the electronegativity of the
fluorine which generates a C-F bond at C5 that cannot be broken
176
R
Mechanism of Thymidylate
Synthase Inhibition by 5-FluorodUMP
• Most normal mammalian cells, requires less
dTMP and so are less sensitive to inhibitors
that inhibit thymidylate synthase or DHF
reductase with exceptions are:
– Bone morrow cells that constitute the bloodforming tissue and much of the immune system
– Intestinal mucosa
– Hair follicles
177
Mechanism of Thymidylate
Synthase Inhibition by 5-FluorodUMP
• 5-FludUMP is irreversible inhibitor of
thymidylate synthase
• It binds to the enzyme and undergoes the 1st
two steps of the normal enzymatic reaction
• In step 3, the enzyme cannot abstract the F
atom as F+ because F is the most
electronegative element, so that enzyme is
bound covalently with F and forming EnzFludUMP-THF ternary complex
178
Enz-FludUMP-THF Ternary
Complex
• The slide shows the Xray structure of this
Enz-FludUMP-THF
ternary complex :
– Its active site region is
shown with helices
(yellow), b-stands
(organe), and other
polypeptides (blue)
– C-5 and C-6 of FludUMP
(green spheres) form
covalent bond (red) with
CH2 group (blue) and S
of Cys (yellow spheres)
179
Orientation of Substrate and
Coenzyme in the Active Site of
Thymidylate Synthase
180
Regeneration of N5, N10Methylenetetrahydrofolate
181
Relationship between THF, Vit B12
and SAM
182
Serine
Hydroxymethyltransferase
183
Serine Hydroxymethyltransferase
(SHMT)
• It catalyzes the reversible conversion of of
Ser to Gly using PLP and THF as coenzymes
• It is unusual enzyme because it utilizes PLP
for C-C bond formation at the oxidation level
of formaldehyde
• It is largely responsible for the provision of
cellular one-carbon methylene
• Because in the reverse reaction it can be
used to generate N5-methylene-THF from Ser
184
SHMT, Cont’d
• It is a part of the a-class of PLP
enzymes
• In the mechanism, Ser is bound to PLP,
and is converted to Gly by cleaving off a
formaldehyde from the Ser side chain,
with the formaldehyde binding to THG,
converting it to N5,N10-methylene THF
185
SHMT, Cont’d
186
SHMT, Cont’d
• SHMT is a common enzyme complex, with
homologous structures present in both
prokaryotes and eukaryotes, including
humans
• These enzymes, though genetically
dissimilar, have matching secondary and
tertiary structures between the subunits of
different species
187
SHMT, Cont’d
• The final enzyme complex of the prokaryotes
and eukaryotes also differs, with the
prokaryotes tending to form tight dimers of
four subunits, while the eukaryotes form
tetramers
• In eukaryotes, SHMT is present in both the
cytosol and the mitochondria
188
SHMT, Cont’d
• The 2 isoenzymes of SHMT exist in cytosolic
and mitochondrial compartments (cSHMT and
mSHMT, respectively) and may have arisen
from a gene duplication event after the
divergence of bacterial and eukaryotic proteins
• Communication between the cytosolic and
mitochondrial compartments in one-carbon
metabolism is achieved using metabolites that
can cross the mitochondrial membrane,
primarily Ser, Gly, and formate
189
SHMT, Cont’d
• Serine is considered the major source of onecarbon units, which are generated through
the production of glycine and 5,10methyleneTHF by both the cytosolic and the
mitochondrial forms of SHMT, but primarily by
mSHMT
• In eukaryotic systems, cSHMT generally
operates in the direction of Ser synthesis,
whereas mSHMT primarily works in the
opposite direction
190
Serine Hydroxymethyltransferase
One subunit of the dimeric enzyme
191
SHMT, Cont’d
192
Mechanism of Serine
Hydroxymethyltransferase
193
Mechanism of Serine
Hydroxymethyltransferase
Lys
Lys
NH
CH
O
O
H
P O
O
H
N C
H
B:
Ser
COO
H2..
N
B:
OH
H2C
HO
N
H
CH3
OH
PLP-Enz
Schiff base
COO
N
H
O
CH2
C
O
P O
O
CH
OH
N
CH3
H
(Aldimine)
PLP-Ser
Schiff base
Electron sink (Stored 1 electron and 1 H+). Source & Where?
194
Mechanism of Serine
Hydroxymethyltransferase, Cont’d
Lys
Lys
+
BH
H2 N
..
H2 N
..
H
H
C
HO
H2C
C
P O
O
O
OH
..
N
CH3
H
Quinonoid
H
N
H2N
N
R
CH
O
COO
N
THF H2O
N
O
C
COO
O
P O
O
CH
OH
N
CH3
H
H
N
N
O
H
CH2NH
THF
B:
Ketimine
195
Electron sink (Stored 1 electron and 1 H+). Source & Where?
Mechanism of Serine
Hydroxymethyltransferase, Cont’d
Lys
H
N
H2N
Lys
H
H2 N
..
N
H2N
R
N
N
H
O
N
H2 N
..
R
N
CH2NH
N
H
O
CH2
C
N
BH+
COO
H
N
CH2NH
B:
CH2
C
H
COO
N
H2N
N
N
O
O
P O
O
CH
O
CH
OH
..
N
H
O
CH3
H
PLP-Ser-Schiff base
P O
O
H
OH
N
H
CH3
O
N
N
CH2
C
N
H2
R
5N, 10N-
methyleneTHF
196
Electron sink (Stored 1 electron and 1 H+). Source & Where?
Mechanism of Serine
Hydroxymethyltransferase, Cont’d
Lys
H
BH+
H
H2 N
..
CH
O
..
Lys
COO
N
O
P
O
C
H2 N
H2O
OH
+
H
BH
CH3
O
H
Quinonoid
O
COO
N
..
N
C
P O
O
CH
O
OH
H
N
H
CH3
B:
H
PLP-Gly-Schiff base
Electron sink (Stored 1 electron and 1 H+). Source & Where?
197
Mechanism of Serine
Hydroxymethyltransferase, Cont’d
Lys
NH
Lys
CH
O
H2 N
..
O
CH
O
O
H
P
O
O
N
O P
O
OH
O
N
CH3
OH
H
CH3
PLP-Enz Schiff base
H
H
C
COO
H2 N
Gly
198