lecture 25 –quiz monday pentose phosphate pathway –this lecture is for wed. –quiz friday on...
TRANSCRIPT
Lecture 25
– Quiz Monday Pentose Phosphate Pathway– This lecture is for WED.– Quiz Friday on TCA cycle– Pyruvate Dehydrogenase Complex (PDC)
3rd stage: carbon-carbon bond cleavage and formation reactions
• Conversion of three C5 sugars to two C6 sugars and one C3 (GAP)
• Catalyzed by two enzymes, transaldolase and transketolase
• Mechanisms generate a stabilized carbanion which interacts with the electrophilic aldehyde center
Transketolase• Transketolase catalyzes the transfer of C2 unit from Xu5P to R5P
resulting in GAP and sedoheptulose-7-phosphate (S7P).• Reaction involves a covalent adduct intermediate between Xu5P and
TPP.• Has a thiamine pyrophosphate cofactor that stabilizes the carbanion
formed on cleavage of the C2-C3 bond of Xu5P.1. The TPP ylid attacks the carbonyl group of Xu5P (C2)2. C2-C3 bond cleavage results in GAP and enzyme bound 2-(1,2-
dihydroxyethyl)-TPP (resonance stabilized carbanion)3. The C2 carbanion attacks the aldehyde carbon of R5P forming an
S7P-TPP adduct.4. TPP is eliminated yielding S7P and the regenerated enzyme.
Thiamine Pyrophosphate (B1)
Thiazolium ring
CH3
CH2
CH3 CH2CH2O-P-P
H
SN
N
N+
very acidic H since the electrons can delocalize into heteroatoms.
Involved in both oxidative and non-oxidative decarboxylation as a carrier of "active" aldehydes.
Pag
e 86
5
Covalent adduct
Carbanion intermediate
Transketolase
• Similar to pyruvate decarboxylase mechanism.• Septulose-7-phosphate (S7P) is the the substrate
for transaldolase.• In a second reaction, a C2 unit is transferred from
a second molecule of Xu5P to E4P (product of transaldolase reaction) to form a molecule of F6P
Transaldolase• Transfers a C3 unit from S7P to GAP yielding erythrose-
4-phosphate (E4P) and F6P.• Reactions occurs by aldol cleavage.• S7P forms a Schiff base with an -amino group of Lys from
the enzyme and carbonyl group of S7P.• Transaldolase and Class I aldolase share a common
reaction mechanism.• Both enzymes are barrel proteins but differ in where
the Lys that forms the Schiff base is located.
Pag
e 86
6• Essential Lys residue forms a Schiff
base with S7P carbonyl group
• A Schiff base-stabilized C3 carbanion is formed in aldol cleavage reaction between C3-C4 yielding E4P.
• The enzyme-bound resonance-stabilized carbanion adds to the carbonyl C of GAP to form F6P.
• The Schiff base hydrolyzes to regenerate the original enzyme and release F6P
Figure 23-31 Summary of carbon skeleton rearrangements in the pentose phosphate
pathway.
Pag
e 86
7
Control of Pentose Phosphate Pathway
1. Principle products are R5P and NADPH.2. Transaldolase and transketolase convert excess R5P
into glycolytic intermediates when NADPH needs are higher than the need for nucleotide biosynthesis.
3. GAP and F6P can be consumed through glycolysis and oxidative phosphorylation.
4. Can also be used for gluconeogenesis to form G6P5. 1 molecule of G6P can be converted via 6 cycles of
PPP and gluconeogenesis to 6 CO2 molecules and generate 12 NADPH molecules.
6. Flux through PPP (rate of NADPH production) is controlled by the glucose-6-phosphate dehydrogense reaction.
7. G6PDH catalyzes the first committed step of the PPP.
Pag
e 76
6
Pyruvate Dehydrogenase Complex (PDC) • In aerobic respiration, NAD+ is recycled by the
electron transport chain.• Also able to utilize energy previously stored as lactate.• Acetyl-CoA is made from pyruvate through oxidative
decarboxylation by a multienzyme complex, pyruvate dehydrogense.
• The general reaction catalyzed:
C-O-
H-C=O
CH3
O
+ NAD+ + CoA-SH
Acetyl-CoA + NADH + CO2
Gº’ = -8 kcal/mol
Pyruvate Dehydrogenase Complex (PDC) • Pyruvate dehydrogenase multienzyme complex (PDC)
consists of three enzymes.• Pyruvate dehydrogenase (E1) form dimers that associate with
E2 at the center of the cubic edges.• Dihydrolipoyl transacetylase (E2) core of the enzyme. In E.
coli has 24 identical subunits with cubic symmetry. • Dihydrolipoyl dehydrogenase (E3) form dimers that are
located on the centers of the cube’s six faces.Gram-negative bacteria have this type. Another type is dodecahedral form found in eukaryotes and
gram-positive bacteria.
Figure 21-4 Structural organization of the E. coli PDC.
Pag
e 76
9
Dihydrolipoyl transacetylase (E2) core
Orange spheres are the 24 pyruvate dehydrogenase (E1) form dimers
Combined a and bDihydrolipoyl transacetylase (E2) core indicated by shaded cube
Purple spheres are the 12 dihydrolipoyl dehydrogenase (E3) subunits also form dimers
Pyruvate Dehydrogenase Complex (PDC) • 5 coenzymes vitaminThiamine pyrophosphate (TPP) thiamineFlavin adenine dinucleotide (FAD) riboflavinCoenzyme A (CoA) pantothenic acidNicotinamide adenine dicleotide (NAD) niacinLipoic acid
• Multienzyme complexes are catalytically efficient and offer advantages over separate enzymes
1. Enzymatic reaction rates are limited by frequency at which enzymes collide with substrates. In a multi-enzyme complex, the distance the substrates must travel is minimized, enhancing rates.
2. Complex formation provides a way of channeling (passing) intermediates between successive enzymes (minimizes side reactions).
3. The reactions may be coordinately controlled.
Figure 21-6 The five reactions of the PDC.
Pag
e 77
0
Pyruvate Dehydrogenase Complex (PDC) • Acetyl-CoA formation occurs over 5 reactions1. Pyruvate dehydrogenase (E1)-decarboxylates pyruvate
using TPP with the intermediate formation of hydroxyethyl-TPP (like pyruvate decarboxylase).
2. Dihydrolipoyl transacetylase (E2)-accepts the hydroxyethyl group from E1.
Thiamine Pyrophosphate (B1)
Thiazolium ring
CH3
CH2
CH3 CH2CH2O-P-P
H
SN
N
N+
very acidic H since the electrons can delocalize into heteroatoms.
Involved in both oxidative and non-oxidative decarboxylation as a carrier of "active" aldehydes.
Mechanism of E1 using TPP
1. Nucleophilic attack by the dipolar cation (ylid) form of TPP on the carbonyl carbon of pyruvate to form a covalent adduct.
2. Loss of carbon dioxide to generate the carbanion adduct in which the thiazolium ring of TPP acts as an electron sink.
3. Pass to next enzyme.
Reaction 1: Pyruvate dehydrogenase (E1) -note how similar to
pyruvate decarboxylase
CH3
P-P-O
S
N
R
(+)
(-)
C-O-
C=O
CH3
O
CH2
CH2
CH3
P-P-O
S
N
R
(+)
CH2
CH2
C-O-
C-OH
CH3
O
CO2TPP (ylid form)
pyruvate
H+
E1
E1
Hydroxyethyl TPP (HETPP)-E1 complex
-CH3
P-P-O
R
CH2
CH2
C-OH
CH3S
N+
Reaction 2: Dihydrolipoyl transacetylase (E2)
S
S
E2
E1
Lipoamide-E2
H+
Hydroxyethyl group carbanion attacks the lipoamide disulfide causing the reduction of the disulfide bond
Hydroxyethyl TPP (HETPP)-E1 complex
CH3
P-P-O
R
CH2
CH2
C-O-H
CH3S
N+
Dihydrolipoyl transacetylase (E2)
HS
S
E2
E1
H+
The TPP is eliminated to form acetyl-dihydrolipoamide and regenerate E1
TPP-E1 complexBack to reaction 1
CH3
P-P-O
R
CH2
CH2
C
CH3
S
N+
Dihydrolipoyl transacetylase (E2)
E1
-
HS
S
E2
Acetyl-dilipoamide-E2
O
C CH3
Reaction 3: Dihydrolipoyl transacetylase (E2)
HS
HS
E2Acetyl-dilipoamide-E2
O
C
CH3
HS
S
E2
O
CoA-SH
CoA-S
dihydrolipamide-E2
E2 catalyzes the transfer of the acetyl group to CoA via a transesterification reaction where the sulfhydryl group of CoA attacks the acetyl group of the acetyl dilipoamide-E2 complex.
+
Reaction 4: Dihydrolipoyl dehydrogenase (E3)
HS
HS
E2E3 is oxidized and catalyzes the oxidation of dihydrolipoamide completing the cycle of E2.
+
FAD
S
SE3 oxidized
FAD
SH
SH
S
S
E2
+E3 reduced
Reaction 5: Dihydrolipoyl dehydrogenase (E3)
E3 is oxidized by the enzyme bound FAD which is reduced to FADH2. This reduces NAD+ to produce NADH.
S
FADH2
S
FAD
SH
SH
NAD+
FAD
S
S
NADH + H+
E3 oxidized
Figure 21-6 The five reactions of the PDC.
Pag
e 77
0
Figure 21-7 Interconversion of lipoamide and dihydrolipoamide.
Pag
e 77
1
Structure of E2
• Consists of several domains• N-terminal Lipoyl domain (80 residues each)-covalently
binds lipoamide• Peripheral subunit-binding domain (35 residues) binds to
E1 and E3• C-terminal catalytic domain (250 residues) catalytic center
and intersubunit binding.• Linked by 20-40 residue Pro/Ala rich segments.
Figure 21-8 Domain structure of the dihydrolipoyl transacetylase (E2) subunit of the PDC.
Pag
e 77
3
The number of lipoyl domains depends on the speciesE. coli, A. vinelandii, n= 3Mammals, n = 2Yeast, n = 1
Figure 21-9 X-Ray structure of a trimer of A. vinelandii dihydrolipoyl transacetylase (E2) catalytic domains. 24
Pag
e 77
3
The N terminal “elbow” extends over neighboring subunit
The CoA and lipoamide bound to enzyme
Structure of E1
• Related to -ketoglutarate dehydrogenase complex and to branched chain keto acid dehydrogenase complex.
• Catalyze the NAD+ linked oxidative decarboxylation of an -keto acid with the transfer of the acyl group to CoA.
• No structure of E1 from PDC has been determined but they make inferences E1 subunits of another keto acid dehydrogenase (P. putida branched-chain-keto acid dehydrogenase, a 2-fold symmetric heterotetramer).
Figure 21-12a X-Ray structure of E1 from P. putida branched-chain -keto acid dehydrogenase. (a)
The 22 heterotetrameric protein.
Pag
e 77
6
Figure 21-12b X-Ray structure of E1 from P. putida branched-chain -keto acid dehydrogenase. (b)
A surface diagram of the active site region.
Pag
e 77
6
Structure of E3
• The reaction is more complex than depicted. • Contains a redox-active disulfide bond that can form a dithiol.• Catalytic mechanism is similar to glutathione reductase.
Figure 21-13a X-Ray structure of dihydrolipoamide dehydrogenase (E3) from P. putida in complex with FAD and NAD+.
(a) The homodimeric enzyme.
Pag
e 77
7
Figure 21-13b X-Ray structure of dihydrolipoamide dehydrogenase (E3) from P. putida in complex with FAD and NAD+.
(b) The enzyme’s active site region.
Pag
e 77
7
Redox active disulfide bridge
Mechanism of E3
• The oxidized enzyme E, which contains the redox-active diulfide bond (S43-S48) binds dihydrolipoamide to form an ES complex.
• His helps with general acid catalysis.• Tyrosine blocks oxidation of FAD by O2 but allows NAD+
access.
•Nucelophillic attack•Proton abstraction yields thiolate ions
Substrate binding
4348
•Substrate thiolate displaces S43 with His as acid catalyst
•S48 forms charge transfer complex with FAD•Lipoamide is released
•Tyr blocks access to FAD.
•NAD+ binds and the Tyr is pushed aside.
•NAD+ is reduced to NADH
•Redox disulfide is reformed.
Pag
e 78
0
•Charge transfer complex-covalent bond formed between Cys48 thiolate and flavin ring. N5 acquires a proton from Cys43.
•Cys43 thiolate nucleophillcially attacks S48 to form the redox active disulfide bond.
•Release of the FADH- anion.
Regulation of PDC
• PDC regulates the entrance of acetyl units derived from carbohydrates into the citric acid cycle.
• The decarboxylation reaction (E1) is irreversible and it is the only pathway for acetyl-CoA synthesis from pyruvate in mammals.
• 2 regulatory systems• Product inhibition by NADH and acetyl-CoA• Covalent modification by phosphorylation/dephosphorylation of the
E1 subunit of pyruvate dehydrogenase.
Figure 21-17a Factors controlling the activity of the PDC.(a) Product inhibition.
Pag
e 78
1
Product inhibition
• NADH and acetyl-CoA compete with NAD+ and CoA for binding sites.
• NADH and acetyl-CoA drive reversible transacetylase (E2) and didhydrolipoyl dehydrogenase (E3) reactions backwards.
Figure 21-17b Factors controlling the activity of the PDC.
(b) Covalent modification in the eukaryotic complex.
Pag
e 78
1
Control by phosporylation/dephosphorylation
• Occurs only in eukaryotic complexes• The E2 subunit has both a pyruvate dehydrogenase
kinase and pyruvate dehydrogenase phosphatase that act to regulate the E1 subunit.
• Kinase inactivates the E1 subunit. Phosphatase activates the subunit.
• Ca2+ is an important secondary messenger, it enhances phosphatase activity.
Pag
e 76
6
Citric acid cycle: 8 enzymes • Oxidize an acetyl group to 2 CO2 molecules and generates 3 NADH, 1 FADH2, and
1 GTP.• Citrate synthase: catalyzes the condensation of acetyl-CoA and oxaloacetate to
yield citrate.• Aconitase: isomerizes citrate to the easily oxidized isocitrate.• Isocitrate dehydrogenase: oxidizes isocitrate to the -keto acid
oxalosuccinate, coupled to NADH formation. Oxalosuccinate is then decarboxylated to form -ketoglutarate. (1st NADH and CO2).
-ketoglutarate dehydrogenase: oxidatively decarboxylates -ketoglutarate to succinyl-CoA. (2nd NADH and CO2).
• Succinyl-CoA synthetase converts succinyl-CoA to succinate. Forms GTP.• Succinate dehydrogenase: catalyzes the oxidation of central single bond of
succinate to a trans double bond, yielding fumarate and FADH2.• Fumarase: catalyzes the hydration of the double bond to produce malate.• Malate dehydrogenase: reforms OAA by oxidizing 2ndary OH group to ketone
(3rd NADH)
Total (PDH and TCA)
3NAD+ + FAD + GDP + Pi + acetyl-CoA
3NADH + FADH2 + GTP + CoA + 2CO2
NAD+ + pyruvate + CoA NADH + acetyl-CoA + CO2
PDH
TCA
Pyruvate4NAD+ FAD GDP + Pi
3CO2
4NADHFADH2
GTP
12ATP
2ATP1ATP
NADH DH
Complex II
Nucleosidediphosphokinase