1. General Metabolism III: Cofactors and Common Themes
Andy Howard Introductory Biochemistry
26 November 2013
Biochemistry: Metabolism III
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2. Common themes in intermediary metabolism
Many of the metabolic systems on which you’ll focus in the spring have common themes uniting them.
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Biochemistry: Metabolism III

3. Metabolism topics Specific systems Cosubstrates Prosthetic groups
SAM, UDP-glucose
Coenzyme A
NAD(P)
Prosthetic groups
Flavins
TPP, PLP
Biotin, THF
Cobalamin, lipoamide
Common themes
Specific systems
Glycolysis and gluconeogenesis
TCA cycle and electron transport
Photosynthesis
Lipid metabolism
Amino acid metabolism
Nucleic Acid metabolism
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Biochemistry: Metabolism III

4. S-adenosylmethionine
Made from methionine and adenosine
Sulfonium group is highly reactive: can donate methyl groups
S-adenosyl-methionine
methionine
ATP
Pi+PPi
This sulfur is chiral; only one enantiomer is active!
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Biochemistry: Metabolism III

5. UDP-glucose Most common donor of glucose
Formed via: Glucose-1P + UTPUDP-glucose + PPi
Reaction driven to right by PPi hydrolysis
UDP-glucose
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Biochemistry: Metabolism III

6. Thioesters: another class of high-energy compounds
Thioesters have similar reactivity as oxygen-acid anhydrides
Thioesters less stable than oxygen esters because the unshared electrons in sulfur are not as delocalized in a thioester as the unshared electrons in an oxygen ester
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Biochemistry: Metabolism III

7. Coenzyme A (G&G p.616)
Reactive portion is free sulfhydryl at one end of the molecule
Can form thioester with acetate, etc.
Pantoate + b-alanine = pantothenate
2-mercapto-ethylamine)
-alanine)
(ADP-3’P)
(Pantoate)
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Biochemistry: Metabolism III

8. NAD+ and NADP+
Net charge isn’t really > 0 ; the + is just a reminder that the nicotinamide ring is positively charged
Most important cosubstrates in oxidation-reduction reactions in aerobic organisms
Nicotinamide adenine dinucleotide phosphate
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Biochemistry: Metabolism III

9. Differences between them
The chemical difference is in the phosphorylation of the 2’ phosphate group of the ribose moiety
The functional difference is that NAD+ is usually associated with catabolic reactions and NADP+ is usually associated with anabolic reactions
Therefore often NAD+ and NADPH are reactants and NADH and NADP+ are products
Exceptions: photosynthesis and ETC!
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Biochemistry: Metabolism III

10. How do we get back to the starting point?
NADH is often oxidized back to NAD+ as part of the electron-transport chain
NADPH is created via photosynthesis
Imbalances can be addressed via NAD Kinase (S.Kawai et al (2005), J.Biol.Chem. 280:39200) and NADP phosphatase
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Biochemistry: Metabolism III

11. Reduced forms of NAD(P)
Reduction occurs on the nicotinamide ring
Ring is no longer net-positive
Ring is still planar but the two hydrogens on the para carbon are not
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Biochemistry: Metabolism III

12. NADPH Provides reducing power for anabolic reactions
Often converting highly oxidized sugar precursors into more reduced molecules
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Biochemistry: Metabolism III

13. FAD and FMN (p. 615) Flavin group based on riboflavin
Flavin mononucleotide
Flavin group based on riboflavin
Alternate participants in redox reactions
Prosthetic groups: tightly but noncovalently bound to their enzymes
That protects against wasteful reoxidation of reduced forms
FADH2 is weaker reducing agent than NADH
These are capable of one-electron oxidations and reductions
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Biochemistry: Metabolism III

14. Flavin adenine dinucleotide (FAD)
(adenosine monophosphate)
FAD and FMN structures
Flavin adenine dinucleotide (FAD)
FAD has an AMP attached P to P
(riboflavin)
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Biochemistry: Metabolism III

15. FMN/FAD redox forms Two-electron version: H+ + :H- transferred
Reaction diagram courtesy of Eric Neeno-Eckwall, Hamline University
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Biochemistry: Metabolism III

16. Thiamine Pyrophosphate (G&G p. 614)
Based on thiamine, vitamin B1
Carboxylases and oxidative decarboxylases use this coenzyme
So do transketolases (move 2 carbons at a time between sugars with keto groups)
Thiazolium ring is reactive center: pKa drops from 15 in H2O to 6 in enzyme
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Biochemistry: Metabolism III

17. TPP
Ylid form of TPP
We already talked about decarboxylations of -ketoacids, e.g. pyruvate + H+  acetaldehyde + CO2
Formation and cleavage of -hydroxylactones & -hydroxyacids: 2 pyruvate + H+  acetolactate + CO2
acetolactate
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Biochemistry: Metabolism III

18. TPP reactions pyrimidine thiazolium
Diagram courtesy of Oklahoma State U. Biochemistry program
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19. Pyridoxal phosphate
PLP is prosthetic group for many amino-acid-related enzymes, including, but not limited to, transaminations
That’s how a lot of -amino acids are synthesized from the corresponding -ketoacids:
α-keto acid 1
α-amino acid 2
α-keto acid 2
α-amino acid 1
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Biochemistry: Metabolism III

20. How PLP functions
Carbonyl group of PLP bound as a Schiff base (imine) to -amino group of lysine at active site
First step is always formation of external aldimine; goes through gem-diamine intermediate to internal aldimine
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Biochemistry: Metabolism III

21. PLP Remember we said it gets used in a lot of transaminations
We should consider its chemistry and its other roles in pathways
To start with: it exists in at least 2 tautomeric forms
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Biochemistry: Metabolism III

22. PLP: Non-transamination reactions
-decarboxylation: -amino acid + H+  CO2 + H3N+-CH2-R
-decarboxylation
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23. Biotin (cf. fig. 22.3) Rarity: vitamin is the prosthetic group
Used in reactions that transfer carboxyl groups
… and in ATP-dependent carboxylations
biotin
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24. Biotin reactivity
Covalently bound to active-site lysines to form species called biocytin
Pyruvate carboxylase is characteristic reaction:
Diagram courtesy University of Virginia Biochemistry
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Biochemistry: Metabolism III

25. Tetrahydrofolate (G&G pp. 894-895)
Primary donor of one-carbon units (formyl, methylene, methyl)
Supplies methyl group for thymidylate
Dihydrofolate reductase (DHFR) is an interesting drug target
Methotrexate as cancer chemotherapeutic: cancer needs more thymidylate than healthy cells
Trimethoprim as antibacterial: Bacterial DHFR is somewhat different from eukaryotic DHFR because bacteria derive DHF from other sources; humans get it from folate
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Biochemistry: Metabolism III

26. THF structure and function
Figure courtesy horticulture program, Purdue
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27. Tetrahydrofolate variations
-2 oxidation state: methyl donor from N5-methyl-THF
0 oxidation state: methylene donor from N5,N10-methylene-THF
+2 oxidation state:
formyl (-CH=O) from N5-formyl-THF and N10-formyl-THF
Formimino (-CH=NH) from N5-formimino-THF
Methenyl (-CH=) from N5,N10-methenyl-THF
See textbook for specifics
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Biochemistry: Metabolism III

28. Cobalamin Largest B vitamin
Structure related to heme but missing one carbon in ring structure
Cobalt bound in core of ring system
Involved in enzymatic rearrangements
Catabolism of odd-chain fatty acids
Methylation of homocysteine
Reductive dehalogenation
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Biochemistry: Metabolism III

29. Adenosyl- Cobalamin Reactive Co-C bond “Missing” carbon
Diagram courtesy of Swiss Food News
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30. Lipoamide (p.616) Protein-bound form of lipoic acid
enzyme
Lipoamide (p.616)
lipoate
Protein-bound form of lipoic acid
Contains five-membered disulfide ring
Covalently bound via amide to protein lysine sidechain
Involved in swinging arm between active sites in multienzyme complexes
Disulfides break periodically
Example: pyruvate dehydrogenase complex
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31. Lipoamide 2e- reduction
thioester starting point
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32. iClicker quiz question 1
Based on what you have learned, would you expect glycogen synthase to be activated or inhibited by phosphorylation?
(a) activated
(b) inhibited
(c) neither
(d) insufficient information to tell
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Biochemistry: Metabolism III

33. iClicker quiz question 2
What would you expect to be the phosphate donor in the NAD kinase reaction?
(a) free phosphate
(b) pyrophosphate
(c) ATP
(d) pyridoxal phosphate
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Biochemistry: Metabolism III

34. iClicker quiz question 3
Which coenzyme would you expect would be required for the reaction oxaloacetate + glutamate  aspartate + a-ketoglutarate? (a) ascorbate (b) PLP (c) thiamine pyrophosphate (d) NAD (e) none of the above
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35. Common themes & specifics
Every major biochemical pathway:
Involves enzymatic activity
Employs cofactors
Includes oxidation-reduction reactions
Contributes to cellular homeostasis
Is subject to control via thermodynamics, localization, transcription, and allostery
We’ll explore the specifics a little now
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36. Glycolysis & gluconeogenesis
Glycolysis: Conversion of sugars (glycogen, glucose, glucose-6-P, fructose, etc.) to pyruvate (catabolic)
Gluconeogenesis: buildup of pyruvate and TCA cycle intermediates to make glucose or glucose-6-P (anabolic)
7 common reactions, 3 divergences due to irreversibility
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37. Glycolysis (table 18.1) Reactants Products Enzyme ΔGo’ ΔG Glucose,ATP
G6P,ADP
Hexokinase
-16.7
-33.9
G6P
F6P
Phosphoglucoisomerase
1.7
-2.9
F6P+ATP
F1,6bisP+ADP
Phosphofructokinase
-14.2
-18.8
F-1,6bisP
DHAP,Glyc3P
Aldolase
23.9
-0.23
DHAP
Glyc3P
Triosephosphate isomerase
7.6
2.4
Glyc3P,NAD,Pi
1,3bisPgl,NADH
Glyc-3-P dehydrogenase
6.3
-1.3
1,3bisPgl,ADP
3-P-glcate,ATP
Phosphoglycerate kinase
-18.9
0.1
3-P-glcate
2-P-glycate
Phosphoglycerate mutase
4.4
0.8
2-P-glcate+H2O
PEP
Enolase
1.8
1.1
PEP+ADP
Pyruvate +ATP
Pyruvate kinase
-25.2
-14.8
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38. Gluconeogenesis (§§22.1-22.2)
Reverses all 7 of the reactions in the previous slide that have ΔG > -5
Differences:
Reactants
Products
Enzyme
ΔGo’ ΔG
Pyruvate+ATP+CO2
Oxalo-acetate
Pyruvate carboxylase
-12?
-4?
OAA
PEP
PEP carboxykinase
+12?
~ 0
F1,6bisP
F6P
Fructose 1,6-bisphosphatase
~ -18
~ -22
G6P
Glucose
Glucose 6-phosphatase
~ -15
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39. TCA cycle (Chapter 19)
Pyruvate has a variety of fates, one of which is to be decarboxylated and oxidized to acetyl CoA via pyruvate dehydrogenase
The acetyl CoA then can condense with oxaloacetate to form a tricarboxylic acid, citrate
Citrate then goes through a series of oxidative transformations that yield 3 NADH, 1 FADH2, and one GTP as it gets converted to oxaloacetate, which can re-enter the cycle
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40. Electron transport (Chapter 20)
The NADH and FADH2 produced in the TCA cycle (or elsewhere) can be reoxidized to NAD and FAD, using O2 as the ultimate electron acceptor
Every NADH reoxidized yields 2.5 ATP
Every FADH2 reoxidized yields 1.5 ATP
The ATP production occurs via a proton pump mechanism in ATP synthase
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Biochemistry: Metabolism III

41. Other sugar pathways (§§22.3-22.6)
Pentose phosphate pathway yields NADPH (which anabolic steps require) and interconverts various sugar phosphates
Glycogen and starch are sugar storage molecules; they can be made and unmade
Glyoxalate pathway (absent in animals) short-circuits the TCA cycle; allows acetyl CoA to serve as a source of sugars
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42. Plant biochemistry (Chapter 21)
Plants, bluegreens, some other organisms can capture light energy and use it to ionize molecules or convert electrons to excited states: photosynthesis
Most of these can grab atmospheric carbon dioxide and incorporate its carbon into organic molecules: RuBisCO
Various interconversions (Calvin cycle) build sugars from there.
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43. Lipid metabolism (Chapters 23 and 24)
Fatty acids are lengthened, 2 C at a time, via malonyl ACP condensing with a growing chain, up to 16 or 18 C, in a single enzymatic complex
Fatty acids are broken down, 2C at a time, via acetyl CoA units
Isoprenoids are built up from mevalonate
Lipid molecules are built up from, and broken down into, FAs and isoprenoid units
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Biochemistry: Metabolism III

44. Amino acid anabolism (Chapter 25, esp. §25.4)
Every amino acid has its own pathway
Many involve transaminations α-ketoacid1 + α-amino acid2  α-aminoacid1 + α-ketoacid2
Most of the precursors are derived from TCA cycle intermediates, with or without phosphorylation
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45. Amino acid catabolism (§25.5)
Again, every story is slightly different
Many of the products lead pyruvate or TCA cycle intermediates; these are considered glucogenic
Some lead to acetyl CoA or acetoacetate and are considered ketogenic; only lys and leu are purely ketogenic.
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46. Nucleic acids (Chapter 26)
Nucleosides synthesized from glutamine, aspartate, glycine, phosphoribosyl pyrophosphate
Interconversions are well-understood
Salvage pathways are surprisingly (to me, at least) important medically
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47. Molecular biology as a biochemical subject (Chapters 28 and 29)
DNA replication, transcription, and translation are proper subjects for biochemical analysis
Enzymes are well-defined and carefully regulated
Some of the enzymes are RNA rather than proteins
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