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Lecture 25 Quiz Monday Pentose Phosphate Pathway

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1 Lecture 25 Quiz Monday Pentose Phosphate Pathway
This lecture is for WED. Quiz Friday on TCA cycle Pyruvate Dehydrogenase Complex (PDC)

2 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

3 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. The TPP ylid attacks the carbonyl group of Xu5P (C2) C2-C3 bond cleavage results in GAP and enzyme bound 2-(1,2-dihydroxyethyl)-TPP (resonance stabilized carbanion) The C2 carbanion attacks the aldehyde carbon of R5P forming an S7P-TPP adduct. TPP is eliminated yielding S7P and the regenerated enzyme.

4 Thiamine Pyrophosphate (B1)
very acidic H since the electrons can delocalize into heteroatoms. H + CH3 CH2 N S N N CH3 CH2CH2O-P-P Thiazolium ring Involved in both oxidative and non-oxidative decarboxylation as a carrier of "active" aldehydes.

5 Carbanion intermediate
Covalent adduct Carbanion intermediate Page 865

6 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

7 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.

8 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 Page 866

9 Figure 23-31 Summary of carbon skeleton rearrangements in the pentose phosphate pathway.
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10 Control of Pentose Phosphate Pathway
Principle products are R5P and NADPH. Transaldolase and transketolase convert excess R5P into glycolytic intermediates when NADPH needs are higher than the need for nucleotide biosynthesis. GAP and F6P can be consumed through glycolysis and oxidative phosphorylation. Can also be used for gluconeogenesis to form G6P 1 molecule of G6P can be converted via 6 cycles of PPP and gluconeogenesis to 6 CO2 molecules and generate 12 NADPH molecules. Flux through PPP (rate of NADPH production) is controlled by the glucose-6-phosphate dehydrogense reaction. G6PDH catalyzes the first committed step of the PPP.

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12 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: O C-O- Acetyl-CoA + NADH + CO2 + NAD+ + CoA-SH H-C=O CH3 Gº’ = -8 kcal/mol

13 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.

14 Figure 21-4 Structural organization of the E. coli PDC.
Purple spheres are the 12 dihydrolipoyl dehydrogenase (E3) subunits also form dimers Orange spheres are the 24 pyruvate dehydrogenase (E1) form dimers a. dihydro Page 769 Dihydrolipoyl transacetylase (E2) core Dihydrolipoyl transacetylase (E2) core indicated by shaded cube Combined a and b

15 Pyruvate Dehydrogenase Complex (PDC)
5 coenzymes vitamin Thiamine pyrophosphate (TPP) thiamine Flavin adenine dinucleotide (FAD) riboflavin Coenzyme A (CoA) pantothenic acid Nicotinamide adenine dicleotide (NAD) niacin Lipoic acid Multienzyme complexes are catalytically efficient and offer advantages over separate enzymes 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. Complex formation provides a way of channeling (passing) intermediates between successive enzymes (minimizes side reactions). The reactions may be coordinately controlled.

16 Figure 21-6 The five reactions of the PDC.
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17 Pyruvate Dehydrogenase Complex (PDC)
Acetyl-CoA formation occurs over 5 reactions Pyruvate dehydrogenase (E1)-decarboxylates pyruvate using TPP with the intermediate formation of hydroxyethyl-TPP (like pyruvate decarboxylase). Dihydrolipoyl transacetylase (E2)-accepts the hydroxyethyl group from E1.

18 Thiamine Pyrophosphate (B1)
very acidic H since the electrons can delocalize into heteroatoms. H + CH3 CH2 N S N N CH3 CH2CH2O-P-P Thiazolium ring Involved in both oxidative and non-oxidative decarboxylation as a carrier of "active" aldehydes.

19 Mechanism of E1 using TPP
Nucleophilic attack by the dipolar cation (ylid) form of TPP on the carbonyl carbon of pyruvate to form a covalent adduct. Loss of carbon dioxide to generate the carbanion adduct in which the thiazolium ring of TPP acts as an electron sink. Pass to next enzyme.

20 Reaction 1: Pyruvate dehydrogenase (E1) -note how similar to pyruvate decarboxylase
C-O- (+) N CH3 C=O (-) H+ CH3 R pyruvate O S E1 (+) C-O- N CH2 CH3 CH2 C-OH P-P-O CH3 S E1 TPP (ylid form) CH2 CO2 CH2 P-P-O

21 Reaction 2: Dihydrolipoyl transacetylase (E2)
CH3 P-P-O R CH2 C-OH S N + - H+ S E2 E1 Lipoamide-E2 Hydroxyethyl TPP (HETPP)-E1 complex Hydroxyethyl group carbanion attacks the lipoamide disulfide causing the reduction of the disulfide bond

22 Dihydrolipoyl transacetylase (E2)
+ S CH3 N H+ C-O-H CH3 E2 HS S CH2 E1 CH2 The TPP is eliminated to form acetyl-dihydrolipoamide and regenerate E1 P-P-O Hydroxyethyl TPP (HETPP)-E1 complex

23 Dihydrolipoyl transacetylase (E2)
CH3 O + C CH3 N S - S HS CH2 E1 CH2 E2 P-P-O TPP-E1 complex Back to reaction 1 Acetyl-dilipoamide-E2

24 Reaction 3: Dihydrolipoyl transacetylase (E2) O
CoA-S C CH3 CH3 O + C S HS CoA-SH HS HS E2 E2 Acetyl-dilipoamide-E2 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.

25 Reaction 4: Dihydrolipoyl dehydrogenase (E3)
FAD FAD S SH S SH E3 reduced E3 oxidized + + S E2 HS E2 E3 is oxidized and catalyzes the oxidation of dihydrolipoamide completing the cycle of E2.

26 Reaction 5: Dihydrolipoyl dehydrogenase (E3)
FAD SH FADH2 S FAD S S S E3 oxidized NADH + H+ NAD+ E3 is oxidized by the enzyme bound FAD which is reduced to FADH2. This reduces NAD+ to produce NADH.

27 Figure 21-6 The five reactions of the PDC.
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28 Figure 21-7 Interconversion of lipoamide and dihydrolipoamide.
Page 771

29 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 residue Pro/Ala rich segments.

30 Figure 21-8 Domain structure of the dihydrolipoyl transacetylase (E2) subunit of the PDC.
The number of lipoyl domains depends on the species E. coli, A. vinelandii, n= 3 Mammals, n = 2 Yeast, n = 1 Page 773

31 Figure 21-9. X-Ray structure of a trimer of A
Figure 21-9 X-Ray structure of a trimer of A. vinelandii dihydrolipoyl transacetylase (E2) catalytic domains. 24 The N terminal “elbow” extends over neighboring subunit The CoA and lipoamide bound to enzyme Page 773

32 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).

33 Figure 21-12a. X-Ray structure of E1 from P
Figure 21-12a X-Ray structure of E1 from P. putida branched-chain a-keto acid dehydrogenase. (a) The a2b2 heterotetrameric protein. The a2B2 heteteromeric protein. Alpha subunits in light blue and gold and the beta subunits in cyan and orange. The TPP bind at the interface of the a and b subunits. Page 776

34 Figure 21-12b. X-Ray structure of E1 from P
Figure 21-12b X-Ray structure of E1 from P. putida branched-chain a-keto acid dehydrogenase. (b) A surface diagram of the active site region. 20 A long channel fromo the surface of the protein to the active site. The lipoyllysyl arm is modeled inof the E2 domain is shown. Page 776

35 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.

36 Figure 21-13a X-Ray structure of dihydrolipoamide dehydrogenase (E3) from P. putida in complex with FAD and NAD+. (a) The homodimeric enzyme. Homodimeric enzyme. One is gray and the other oclored by domain. FAD binding domain is shown in lavender andd the NAD+ domain is shown in cyan. Central domain is shown in yellow. NAD+ is shown in green and the FAD in yellow. Page 777

37 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. Redox active disulfide bridge Enzyme active site region. The redox portions of the bound NAD+ and FAD cofactors are shown and Tyr181 is shown. Page 777

38 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.

39 48 43 Substrate binding Nucelophillic attack Redox disulfide is reformed. Proton abstraction yields thiolate ions NAD+ is reduced to NADH Substrate thiolate displaces S43 with His as acid catalyst NAD+ binds and the Tyr is pushed aside. S48 forms charge transfer complex with FAD Lipoamide is released Tyr blocks access to FAD.

40 Release of the FADH- anion.
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. Page 780 Release of the FADH- anion.

41 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.

42 Figure 21-17a. Factors controlling the activity of the PDC
Figure 21-17a Factors controlling the activity of the PDC. (a) Product inhibition. Page 781

43 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.

44 Figure 21-17b. Factors controlling the activity of the PDC
Figure 21-17b Factors controlling the activity of the PDC. (b) Covalent modification in the eukaryotic complex. Page 781

45 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.

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47 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)

48 Total (PDH and TCA) PDH NAD+ + pyruvate + CoA NADH + acetyl-CoA + CO2
3NAD+ + FAD + GDP + Pi + acetyl-CoA 3NADH + FADH2 + GTP + CoA + 2CO2 Pyruvate 4NAD+ FAD GDP + Pi 3CO2 4NADH FADH2 GTP NADH DH 12ATP Complex II 2ATP 1ATP Nucleoside diphosphokinase

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