1 part 2 coenzymes-dependent enzyme mechanism professor a. s. alhomida disclaimer the texts, tables...
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3 Experimental Evidences for Stereochemistry Hydride Transfer is StereospecificTRANSCRIPT
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Part 2 Coenzymes-Dependent Enzyme
MechanismProfessor A. S. Alhomida
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
King Saud UniversityCollege of Science
Department of Biochemistry
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Experimental Evidences for Stereochemistry Hydride Transfer is Stereospecific
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NR
HCONH2
+ CCHH
HOH
D
D
yeastAD
NR
CONH2
DH
CCHH
HD
O+
NR
CONH2
DH
CCHH
HH
O+
yeastAD
NR
HCONH2
+ CCHH
HOH
H
D
*
CCHH
HOH
H
DCCH
H
HO
H
DSO
O
HOCCH
H
HOH
D
H* stereochemistry
inverted
CCHH
HOH
D
H+N
R
HCONH2 yeast
AD
NR
CONH2
HHCCH
H
HD
O+
*
*
50: 50
100%
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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:
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• 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%)
Stereospecificity of ADH
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• 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
Stereospecificity of ADH
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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
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ADH with bound NAD+
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ADH with bound NAD+ and Trifluoroenanol (TFE)
• The zinc (orange) is coordinated to His-67, Cys-174, 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
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Conformation of the nicotinamide cofactor determines if thepro-R or the pro-S hydrogen is transferred from the cofactor
ORO
HO OH
N
O
H2N
HR
HS
no free rotation
Syn
ORO
HO OH
N
HS
HRCONH2
Anti
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Conformation of NAD+ cofactors
NAD+ from Lactate Dehydrogenase (Pro-R specific)
NAD+ from Glyceraldehyde-3-phosphate Dehydrogenase (Pro-S specific)
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Stereospecificity at C-4 for some NAD-Dependent DH
Enzyme Nucleotide C-4 pro H ProductADH NAD HR Acetaldehyde
UDP-glucose DH
NAD HS UDP-glucuronate
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+
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Mechanism of Alcohol Dehydrogenase
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Mechanism of Alcohol Dehydrogenase
Zn2+ Ser
O
H
N
NH
OH
CH
H
CH3
B:
N
R
NH2
O
His
NAD+
Ethanol
+
Cys-174
Cys-46 His-67
-48
-61
Electron sink (Stored 2 electrons and one H+). Source & Where?
The Zn2+ increases the acidity of the alcohol, but is not involved in the redox reaction
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H2O
CH3C
Alcohol Dehydrogeanse
Zn2+ Ser
OH
OH
H
N
N
H
His
O
H
NADH
+N
NH2
R
HH
..
O
Acetaldehyde
BH+
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Glyceraldehyde 3-phosphate Dehydrogenase
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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
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Glyceraldehyde 3-phosphate Dehydrogenase
• In addition to its role in glycolysis, GAPDH is known to be involved in several other non-glycolytic functions as well (e.g. apoptosis, DNA repair and regulation of histone gene expression
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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
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Glyceraldehyde 3-phosphate Dehydrogenase
• It is a medium-sized tetrameric enzyme • It resembles several other tetrameric NAD-
dependent 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
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Structure of GAPDH shows the tetramer subunits
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GAPDH Reaction
G˚ = 6.3 kj/mol
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Experimental Evidence for Pi Role
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What is the role of Pi?
GAPDHNo product
1. If run with catalytic amount of enzyme and omit Pi from the mixture of the reaction, no product is formed
CH
OHC
CH2OP
H
O
O2
3
+ NAD+
3-PG
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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
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Experimental Evidence for Pi Role, Cont’d
4. At this point, only small amount of the available free NAD+ and substrate had been consumed
GAPDHC
H
OHC
CH2OP
H
O
3-PG
O2
3
+ NAD+
C
OHC
CH2OP
H
O
1, 3 bPG
O2
3
+ NADH
OPO32
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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
C
H
OHC
CH2OP
H
O
3-PG
O2
3
+ Pi
C
OHC
CH2OP
H
O
1, 3 bPG
O2
3
OPO32
NAD+NADH
PAPDH
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Results of the Experimental Evidence for Pi Role
Time (min)
Abs
orba
nce
(340
nm
)
Add Pi
1st burst of NADH formation
2nd burst of NADH formation
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GAPDH Reaction, (Cont’d)
The +ve on the NAD+ may help polarize the thioester to facilitate the attack by Pi
Oxidation step
Phosphorylation step
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Structure of GAPDH shows the active site includes Cys, His residue adjacent to a bound NAD+
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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
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Glyceraldehyde 3-phosphate Dehydrogenase, Cont’d
• GAPDH reaction is the site of action of arsenate (AsO4
3-), an anion analogous to phosphate
• Arsenate is an effective substrate in this reaction, forming 1-arseno-3-phosphoglycerate (1-Ars-PG), but acyl arsenates are quite unstable and are rapidly hydrolyzed
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• 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
Glyceraldehyde 3-phosphate Dehydrogenase, Cont’d
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Mechanism of Glyceraldehyde 3-phosphate Dehydrogenase
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Mechanism of Glyceraldehyde 3-phosphate Dehydrogenase
Electron sink (Stored 2 electrons and one H+). Source & Where?
HisSHCys C
OH
OHC
CH2OP
H
GAP
B:H
N
NH
HisCys C O
H
OHC
CH2OP
H
S H
Tertahedral Intermediate
N
NH2+
O
NAD+
N
NB:
BH
R
Cys increases the acidity of GAP, but is not involved in the redox reaction
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P OHO
OH
O
NADH + H
HisCys CO
OHC
CH2OP
H
S
P OH
OH
O
O
BH
NN
H
BH
Acyl-thioester Intermediate
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HisCys CO
OHC
CH2OP
H
S
P O
OH
O
O
BH
NN
H
BH
3-Phosphoglyceryl-Enz Intermediate1. 3-PG-Enz is a
destabilized acylthioester
2. It undergoes 3-phosphoglyceryl transfer to one of the oxygen of Pi bound at the active site
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CO
OHC
CH2OP
H
P O
O
O
O
1, 3 bPG
His
NN
Cys SH
BH
High energy compound (Acylphosphate
has G˚ = - 49.3 kj/mol)
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Mechanism of Energy Coupling
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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
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3. The oxidation reaction is favored by the deprotonation of the hemithioacetal by His-176
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
Mechanism of Energy Coupling, Cont’d
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Free Energy Profiles for GAP Oxidation
• (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
Mechanism of Energy Coupling, Cont’d
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Riboflavin Coenzymes
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Riboflavin in Foods
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Riboflavins
• Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are derived from riboflavin (Vit B2)
• Flavin coenzymes are involved in oxidation-reduction reactions for many enzymes (flavoenzymes or flavoproteins)
• FAD and FMN catalyze one or two electron transfers
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Riboflavin and its coenzymes
(a) Riboflavin, (b) FMN (black), FAD (black/blue)
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Reduction, reoxidation of FMN or FAD
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Biosynthesis of FAD
Riboflain (Vit B2)
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N
N
NH
N O
O
OH
OH
HO
OH
1
5
10
4a
5'
N
N
NH
N O
O
O
OH
HO
OH
O-
P
O
P OO
O-O
HO
N
OH
O
N
N
N
NH2
N
N
NH
N O
O
O
OH
HO
OH
O-
P
O
O-
Riboflavin Flavin Mononucleotide (FMN) Flavin Adenine Diphosphate (FAD)
N
N
NH
N
CH3
O
OLumiflavin
NH
HN
NH
N NH2
O
OH
HO H
5,6,7,8-tetrahydrobiopterin
N
NH
N O
O
OR
OH
HO
OH
5-Deazaflavins(more closely related to NAD)
Flavin Coenzyme: Vitamin B2, one- and two-electron transfer
tricyclic flavin ring system: isoalloxazine
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One and Two Electron Reaction of Flavin
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Reaction of Flavin with O2
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1. Oxidases: reduced flavin cofactor re-oxidized directly by O2
2. Dehydrogenase: reduced flavin re-oxidized by another group, i.e.,
3. Mixed Function Oxidase: reduced flavin reacts with O2 to give aflavin-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):
O2
R-S-S-R
FlH-red2 R-SH
FloxH2O2
R-S-S-R
Classifications of Flavoenzymes
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Oxygen:
O O O O3 1
triplet singlet
spin: s = 2n +1spin of an electron = ± 1/2
For oxidases and mixed function oxidases
For mixed function oxidases, the flavin 4a-hydroperoxide is theoxygen-transfer (oxidizing) agent
pKa H2O2 ~ 11.6
O O3
NH
N
NH
NR
O
O
+
electron-tranfer
NH
N
NH
NR
O
O
O O+1
spininversion
NH
N
NH
NR
O
O
OO
H+
N
N
NH
NR
O
O
OHO
Flavin-4a-hydroperoxide
for oxidases
HB:H+
N
N
NH
NR
O
O+ H2O2
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For mixed function oxidases (monooxygenases), the oxidized flavin Reducing agent is often NAD(P)H and is usually supplieds a separate enzyme
NAD
FAD
N
N
NH
NR
O
O
NH
N
NH
NR
O
O
NAD(P)H NAD(P)
N
NHN
NO
O
R
NicotinamideE°'~ -0.32 V
FlavinE°'~ -0.20 V
N
HH
H2NOC
R
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• 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)
Mechanism of Flaovenzymes
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Carbanion Mechanism
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D-Amino Acid Oxidase
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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 -imino acids and, after subsequent hydrolysis, -keto acids and ammonia
• It spreads from yeasts to human and it is not present in bacteria nor in plants
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• Recently mammalian D-amino acid has been connected to the brain D-Ser metabolism and to the regulation of the glutamatergic neurotransmission
D-Amino Acid Oxidase, Cont’d
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Structure of D-Amino Acid Oxidase
Human DAAOYeast DAAO
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• D-Ala is located above the reduced flavin Re-side
• (- - -) lines denote hydrogen bonds involved in substrate fixation
Structure of D-Amino Acid Oxidase, Cont’d
Yeast DAAO
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Stabilization of the Negative Charge in the Active Site by Arg-285
Yeast DAAO
DAAO + D-Ala
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Potential of the DAAO-based Selection system
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Potential of the DAAO-based Selection System
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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)
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• (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
Potential of DAAO-based Selection System, Cont’d
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L-Alanine D-Alanine
Structure of D- and L-Alanine
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Structure of D- and L-Alanine (Cont’d)
D-AlanineL-Alanine
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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 N-nucleophile mechanism but not by C-nucleophile mechanism because X-ray structure shows no active site base
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• 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
D-Amino Acid Oxidase,Cont’d
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• 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
D-Amino Acid Oxidase, Cont’d
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Reaction of D-Amino Acid Oxidase
D-Alanine
C
O
OH CNH3
HCH3
C
O
OH CNH2
CH3
C
O
OH CNH3
HCH3
Enz-FAD Enz-FADH2
Enz-FAD
ReoxStep
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Reaction of D-AA Oxidase, Cont’d
Hydrogen peroxide
O2
H2O2
+C
O
OH CNH2
CH3
EnzFAD
Enz-FAD
H2O2
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Reaction of D-AA Oxidase, Cont’d
Nonenzymatic Rnx
Pyruvate
Pyruvate imine
C
O
OH CNH2
CH3
H2O
NH4
C
O
OH CCH3
O
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Mechanism of D-Amino Acid Oxidase
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C-nucleophile mechanism. Amino acid enolate adds to the flavin 4a-position. The flavin then acts as a leaving group
N
N
NH
NR
O
OR CO2H
H2N H :B
R CO2H
H2N
H+ NH
N
NH
NR
O
OR CO2HH2N
NH
N
NH
NR
O
OR CO2H
H2NH2O
R CO2H
ONH4++
pKa~ 25
X-ray structure shows NO active site base
Mechanism of D-Amino Acid Oxidase
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Mechanism of D-Amino Acid Oxidase
(Hydride Transfer Mechanism)N-Nucleophile Mechanism
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N
NNH
NH3C
H3CO
O
R
BH+
Arg-285C
Ser-335
C COO
CH3
H
NH
H
HB:
C COO
CH3
N
H
H
N
NNH
NH3C
H3CO
O
R H
H
O2O O
B:
BH+
OH
NH2H2N
D-Amino Acid
FAD FADH2
Electron sink (Stored 2 electrons and 2 H+). Source & Where?
Pyruvate imine
Redox step
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Mechanism of D-AA Oxidase, Cont’d
FAD
Hydrogen peroxide
N
NNH
NH3C
H3CO
O
R
H
O OHB:
BH+
H2O2
N
NNH
NH3C
H3CO
O
R
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Mechanism of D-AA Oxidase, Cont’d
C
O
OH CNH2
CH3
H2O
NH4
C
O
OH CCH3
O
Pyruvate imine
Non-enzymatic Rxn
Pyruvate
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p-hydroxybenzoate Hydroxylase
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• 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
p-hydroxybenzoate Hydroxylase
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• 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
p-hydroxybenzoate Hydroxylase, Cont’d
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• A chain of H-bonds can connect p-hydroxy-benzoate 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
p-hydroxybenzoate Hydroxylase, Cont’d
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• 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)
Stabilization of Transition State of p-hydroxybenzoate Hydroxylase
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Reaction of p-hydroxybenzoate Hydroxylase
COOHO
NADPHNADP
O2 H2O COOHO
HOEnz-FADH2 Enz-FAD
+ +
4-hydroxybenzoate 3,4-dihydroxybenzoate
Reductase
Hydroxylase
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Reaction Steps of p-hydroxybenzoate Hydroxylase
Reduced form
Reduced form
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Mechanism of p-hydroxybenzoate Hydroxylase
(Monooxygenase)
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N
NNH
NH3C
H3CO
O
R
N
NNH
NH3C
H3CO
O
R H
H
O2 O O
B:
BH+H
H
FADH2
Mechanism of p-hydroxybenzoate Hydroxylase (Monooxygenase)
Electron sink (Stored 2 electrons and 2 H+). Source & Where?
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Hydroxybenzoate
Mechanism of p-HB Hydroxylase (Cont’d)
Electrophilic oxygenN
NNH
NH3C
H3CO
O
R
H O OH
BH+
CCO
O H
N
NNH
NH3C
H3CO
O
R
H O
CCO
OHO
H
H
B:
BH+
B:
BH+
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NADPH
FAD
Electron sink (Stored 2 electrons and 2 H+). Source & Where?
N
NNH
NH3C
H3CO
O
R
NADPH
HO
COO
HO
N
R
NH2
OH H
..
BH+
+H2O
Mechanism of p-HB Hydroxylase, Cont’d
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N
NNH
NH3C
H3CO
O
R
H
H
N
NH2
O
+
R
Reductase
NADP+
FADH2
Mechanism of p-hydroxybenzoate Hydroxylase, Cont’d
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Glutathione Reductase
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Biosynthesis of Glutathione
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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
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• 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 NADPH-binding domains that composed of a babab
Glutathione Reductase
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• 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
Glutathione Reductase
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• 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
Glutathione Reductase
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Glutathione Reductase Catalytic Cycle
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Reaction of Glutathione Reductase
GS-SG
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Mechanism of Glutathione Reductase
(Indirect-NAD-Hydride Transfer)
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Mechanism of Glutathione Reductase(Indirect-NAD-Hydride Transfer)
His
SS
Cys-58 Cys-63
N
NNH
NH3C
H3CO
O
R
N
R
NH2
OH H
..
3 BH+
N
NH
B:
SS
N
NNH
NH3C
H3CO
O
R
H3
H
NADP+
N
N
H
FAD FADH2
NADPH Electron sink (Stored 2 electrons and 2 H+). Source & Where?
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Mechanism of Glutathione Reductase, Cont’d
BH+
SS
N
NNH
NH3C
H3CO
O
R H
3H
N
NGSSG
G S GS
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Mechanism of Glutathione Reductase, Cont’d
B:
SS
N
NNH
NH3C
H3CO
O
R H
N
N
H
G S
GS3H
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Mechanism of Glutathione Reductase, Cont’d
BH+
SS
N
NNH
NH3C
H3CO
O
R
N
N
G S
H
GSH
SS
N
NNH
NH3C
H3CO
O
R
N
N
H
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NAD/NADH versus FAD/FADH2
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NAD/NADH vs FAD/FADH2
• FAD binds tightly to the enzymes (sometimes covalently attached to Cys or His through C-8a) 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
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• 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
NAD/NADH vs FAD/FADH2, Cont’d
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• 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)
NAD/NADH vs FAD/FADH2, Cont’d
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• 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
NAD/NADH vs FAD/FADH2, Cont’d
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• 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
NAD/NADH vs FAD/FADH2, Cont’d
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• 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
NAD/NADH vs FAD/FADH2, Cont’d
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Pterin Coenzyme
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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)
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Pterin, Folate and Tetrahydrofolate (THF or FH4)
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Biosynthesis of Tetrahydrobiopterin (THB)
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Biosynthesis of THB, Cont’d
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Biosynthesis of THB, Cont’d
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• THB is the coenzyme for Phe 4-hydroxylase (PAH), Tyr 3-hydroxylase, andTrp 5-hydroxylase; 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
Structure of THB
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Phenylalanine Hydroxylase
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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
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• PAH includes a non-heme 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
His
His Glu
7,8-dihydrobiopterin
Phenylalanine Hydroxylase PDB 1DMW
Structure of PAH, Cont’d
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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
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• 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
Structure of PAH, Cont’d
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Structure of PAH, Cont’d
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Structure of PAH, Cont’d
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Reactions of PAH and THF
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• 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
Deficiency of PAH
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• 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
Deficiency of PAH, Cont’d
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• 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
Experimental Evidence for 1,2-Shift Mechanism
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D
CO2-
NH3 HO
CO2-
NH3
D
~50% D- incorporation
CO2-
NH3HODB:
CO2-
NH3HOThis mechanism would require NO D in the product
Electrophilic substitution analogous top-hydroxybenzoatehydroxylase
Experimental Evidence for 1,2-Shift Mechanism (Cont’d)
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D
CO2-
NH3NH
HN N
NH
O
NH2OH
OH
H
OHO
+CO2
-
NH3O
D
H
CO2-
NH3O
D
HH
B
+
1,2-hydrideshift
CO2-
NH3HOD H
CO2-
NH3HOH (D)
~50% D- incorporation
Experimental Evidence for 1,2-Shift Mechanism, Cont’d
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Mechanism of PAH(Mono-oxygenase)
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Mechanism of PAH(Mono-oxygenase)
H2N
OH
H
H
CH C H CH3
OHOH
N
N N
N
OO
B:
H2N
O
H
H
N
N N
N
OO
His-290
Glu-330
His-285Fe2+
H2O H2O
H2O
RTHB
Pterin hydroperoxideElectron sink (Stored 1 electron and H+). Source & Where?
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Mechanism of PAH, Cont’d
H2N
O
H
H
N
N N
N
O
H2O
Fe2+
H2O
O
RH2OO
H
H
N
N N
N
OHR
NH
H B:
BH+
H2O
Fe4+
H2O
O2
+ HOHPterin-4a-carbinolamine
Oxyferry
Electron sink (Stored 1 electron and 1 H+). Source & Where?
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Mechanism of PAH, Cont’d
Fe4+
O
RH3H2O
Fe4+
H2O
O2
Phe
B:
O
H
N
N N
N
NH
H
BH+ R
Oxyferry
Phe
Dehydrogenase
13
2
Oxyferry
Electron sink (Stored 1 electron and 1 H+). Source & Where?
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H2N
OH
H
H
CH C H CH3
OHOH
N
N N
N
H3
NADPH3
NADP+
Mechanism of PAH, Cont’d
1
DHB reductase
THB
Electron sink (Stored 2 electrons and 1 H+). Source & Where?
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2
DHB reductase
THB
O
H
N
N N
N
NH
R
NADP+
NADPH
HB:
BH+
O
H
N
N N
N
NH
R
H
H
H2N
OH
H
H
CH C H CH3
OHOH
N
N N
N
DHB
Electron sink (Stored 2 electrons and H+). Source & Where?
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RH3
HO
RH3
H
OH
H
RH3
H
OH
RH3
H
O
R
3
OH
H
TyrH
Carbonion at C3
Hydrogen ion migration
Resonance-stabilized oxonium ion
Epoxide
3
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Tetrahydrofolate (THF)
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• 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
Biosynthesis and Absorption of THF
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Folic Acid (Vitamin B9)We cannot synthesize
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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)
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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
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• 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
THF, Cont’d
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• 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
THF, Cont’d
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Formation of THF from folate
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• One-carbon derivatives of THF
• Note: these are positions 5,6,7,9 and 10
Continued next slide
Different Forms of THF
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THF, Vitamin B12 and SAM
THF THF-CDHF
dUMPdTMPNADPHNADP+ A
Purine precursors
Purines (C2 and C8)
B
GlySer C
B12
B12-CH3
Homocysteine
Meth
SAMSAH
CH3
NorepinephrineGuanidinoacetateNucleotidesPhosphatidylethanolamineAcetylserotonin
Methyated nucleotidesCreatine
PhosphatidylcholineMelatonin
Epinephrine
D
Glucose
Ser
Gly1
His
Formimino-GluGlu NH4
+
2NH4
+Gly
3CO2 +
+
Epinephrine
Formaldehyde4 Trp
Formate
5
Recipients of carbon
Sources of carbon(1 - 5)
(A - D)
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THF in the Metabolism of One-Carbon Units
methyl
methylene
formyl
Single carbon groups can be carried on N-5,N-10, or bridged between N-5 and N-10
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
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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
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• 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, Cont’d
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• 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
Folate Deficiencies, Cont’d
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Homocysteine
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Homocysteine
Homocysteinuria• Rare; deficiency of cystathionine b-synthase• Dislocated optical lenses• Mental retardation• Osteoporosis• Cardiovascular disease death
High blood levels of homocysteine associated withcardiovascular disease
• May be related to dietary folate deficiency• Folate enhances conversion of homocysteine to methionine
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Enzyme Deficiencies Causing Homocystinuria
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Thymidylate Synthase
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Reaction of Thymidylate Synthase
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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
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Thymidylate Synthase, Cont’d
• Synthesis of dTMP from dUMP is catalyzed by thymidylate synthase
• The 5-CH3 group is ultimately derived from the -carbon of serine
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• 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'-deoxy-5-fluorouridylate, a potent inhibitor of dTMP synthase
• 5-Flu is used as a chemotherapeutic agent in the treatment of cancer
Thymidylate Synthase, Cont’d
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• Similarly, 5-fluorocytosine is used as an antifungal drug because fungi, unlike mammals, can convert it to 2'-deoxy-5-fluorouridylate
• 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
Thymidylate Synthase, Cont’d
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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
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• Sulfa drugs, or sulfonamides, owe their antibiotic properties to their similarity to p-aminobenzoate (PABA), an important precursor in folate synthesis
Thymidylate Synthase, Cont’d
• Sulfonamides block folate formation by competing with PABA
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Human thymidylate synthase
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Mechanism of Thymidylate Synthase
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Mechanism of Thymidylate Synthase
N5, N10-Methylene-THF
dUMP
Electron sink (Stored 2 electrons and 2 H+). Source & Where?
Cys
N
N
N
N
H2N
O
H
HCH2
N RH2C
R..BH+
S
N
N
N
N
H2N
O
H
HCH2
N R
R
H2C
H
O
HH
OH H
HH
N
NH
O
O
OCH2P
O
OHO
H
Cys
S
HB:
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Mechanism of Thymidylate Synthase, Cont’d
Electron sink (Stored 2 electrons and 2 H+). Source & Where?
N
N
N
N
H2N
O
H
HCH2
N R
R
H2CH
O
HH
OH H
HH
N
NH
O
O
OCH2PO
OHO
Cys
S
N
N
N
N
H2N
O
H
H CH2
NH2C
H
OCH2PO
OHO
Cys
S
BH+
H
B:
O
HH
OH H
HH
N
NH
O
O
R
R
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Mechanism of Thymidylate Synthase, Cont’d
N
N
N
N
H2N
O
H
HCH2
H2C
OCH2PO
OHO
Cys
S
R
RN
H
H
O
HH
OH H
HH
N
NH
O
O
B:H
BH+
Cys
S
H
N
N
N
N
H2N
O
H
HCH2 R
RN
H
+OCH2P
O
OHO O
HH
OH H
HH
N
NH
O
OH3C
dTMP
Electron sink (Stored 2 electrons and 2 H+). Source & Where?
7, 8-DHF
Hydrogen transfer
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Mechanism of Thymidylate Synthase Inhibition by 5-
Fluoro-dUMP
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Mechanism of Thymidylate Synthase Inhibition by 5-Fluoro-dUMP
N5, N10-Methylene-THF
FdUMP
Cys
N
N
N
N
H2N
O
H
HCH2
N RH2C
R..BH+
S
N
N
N
N
H2N
O
H
HCH2
N R
R
H2C
H
O
HH
OH H
HH
N
NH
O
O
OCH2P
O
OHO
H
Cys
S
HB:
F
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N
N
N
N
H2N
O
H
HCH2
N R
R
H2C
H
O
HH
OH H
HH
N
NH
O
O
OCH2PO
OHO
Cys
S
N
N
N
N
H2N
O
H
H CH2
NH2C
H
OCH2PO
OHO
Cys
S
B:
O
HH
OH H
HH
N
NH
O
O
R
R
F F
X
Mechanism of Thymidylate Synthase Inhibition by 5-Fluoro-
dUMP
Inhibition by Flu-dUMP results from the electronegativity of the fluorine which generates a C-F bond at C5 that cannot be broken
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• 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 blood-
forming tissue and much of the immune system– Intestinal mucosa– Hair follicles
Mechanism of Thymidylate Synthase Inhibition by 5-Fluoro-
dUMP
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• 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 Enz-FludUMP-THF ternary complex
Mechanism of Thymidylate Synthase Inhibition by 5-Fluoro-
dUMP
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Enz-FludUMP-THF Ternary Complex
• The slide shows the X-ray structure of this Enz-FludUMP-THF ternary complex :– Its active site region is
shown with helices (yellow), -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)
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Orientation of Substrate and Coenzyme in the Active Site of
Thymidylate Synthase
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Regeneration of N5, N10-Methylenetetrahydrofolate
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Relationship between THF, Vit B12 and SAM
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Serine Hydroxymethyltransferase
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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
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• It is a part of the -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
SHMT, Cont’d
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SHMT, Cont’d
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• 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
SHMT, Cont’d
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• 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
SHMT, Cont’d
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• 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
SHMT, Cont’d
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• Serine is considered the major source of one-carbon units, which are generated through the production of glycine and 5,10-methyleneTHF 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
SHMT, Cont’d
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Serine Hydroxymethyltransferase
One subunit of the dimeric enzyme
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SHMT, Cont’d
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Mechanism of Serine Hydroxymethyltransferase
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Mechanism of Serine Hydroxymethyltransferase
N
OH
CH3
H
C
OP
O
OO
LysNH
H
CN COO
CH2
HH
H
OHB:N
OH
CH3
H
C
OP
O
OO
LysNH2
H
C COO
H
H2CHO
N
B:..
Electron sink (Stored 1 electron and 1 H+). Source & Where?
PLP-Enz Schiff base
(Aldimine)Ser PLP-Ser Schiff base
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Mechanism of Serine Hydroxymethyltransferase, Cont’d
N
OH
CH3
H
C
OP
O
OO
LysNH2
H
C COOH2CHO
N
BH+
..
..
N
OH
CH3
H
C
OP
O
OO
Lys
NH2
H
C COO
N
..
C
H H
THF H2OR
N
N
N
N
H2N
OCH2NH
H
H
H
THF
B:
Quinonoid
Electron sink (Stored 1 electron and 1 H+). Source & Where?
Ketimine
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Mechanism of Serine Hydroxymethyltransferase, Cont’d
N
OH
CH3
H
C
OP
O
OO
LysNH2
H
C COO
N
..
C
RN
N
N
N
H2N
OCH2NH
H
..
H2 BH+
N
OH
CH3
C
OP
O
O
NH2
H
C COO
N
..
C
N
N
N
N
H2N
OCH2NH
H
H2
Lys
H
HH
O
R
B:H H
C
R
N
N
N
N
H2N
O
H2
H
HC
H2
N
PLP-Ser-Schiff base
Electron sink (Stored 1 electron and 1 H+). Source & Where?
5N, 10N-methylene-THF
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197
Mechanism of Serine Hydroxymethyltransferase, Cont’d
Quinonoid
Electron sink (Stored 1 electron and 1 H+). Source & Where?
PLP-Gly-Schiff base
N
OH
CH3
H
C
OP
O
O
LysNH2
H
C COO
N
..
..
H
BH+
H2O
N
OH
CH3
H
C
OP
O
OO
Lys
NH2
H
C COO
N
..
H
H
OH
B:
BH+
H
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198
Mechanism of Serine Hydroxymethyltransferase, Cont’d
Gly
PLP-Enz Schiff base
H2
C COO
N
H
H N
OH
CH3
H
C
OP
O
OO
LysNH2
H
..O
N
OH
CH3
H
C
OP
O
OO
LysNH
H