1
Q
What is the pentose phosphate pathway (PPP)?
A
Metabolic pathway in which glucose-6-P is oxidized then decarboxylated, generating ribulose-5-P and NADPH
2
Q
Where does the PPP occur?
A
In the cytoplasm
3
Q
What are the important functions of the PPP?
A
- Reduce 2 molecules of NADP+ to NADPH for each molecule of glucose-6-P that is oxidatively decarboxylated to ribulose-5-P
- Produce ribose-5-P from glucose-6-P
4
Q
What does NADPH function as?
A
A strong reductant (electron donor)
5
Q
What is the [NAD+]/[NADH] in liver cells?
A
~1000
6
Q
What is the [NADP+]/[NADPH] in liver cells?
A
0.01
7
Q
What are the 2 phases of the PPP?
A
- Oxidative
- Non-oxidative
8
Q
Flux through both phases of the PPP is regulated to meet what 3 metabolic states?
A
- If increased NADPH is required
- If nucleotide pools need to be replenished
- If ATP levels in the cell are low
9
Q
What happens in the PPP if increased NADPH is required?
A
Fructose-6-P and glyceraldehyde-3-P are used to resynthesize glucose-6-P, thereby maintaining flux through the oxidative phase
10
Q
What happens if nucleotide pools need to be replenished?
A
The bulk of ribulose-5-P is converted to ribose-5-P, stimulating nucleotide biosynthesis
11
Q
What does the PPP accomplish for the cell? (i.e. what are its functions)
A
- Generate NADPH (needed in lots of pathways and to detox ROS)
- Produce ribose-5-P for nucleotide synthesis
- Regenerate glucose-6-P (to maintain NADPH production)
12
Q
What is the over all net reaction of the PPP?
A
6 Glucose-6-P + 12 NADP+ + 6 H2O –>
4 Fructose-6-P + 2 Glyceraldehyde-3-P + 12 NADPH + 12 H+ + 6 CO2
13
Q
What are the key enzymes in the PPP?
A
- G6PD
- Transketolase and transaldolase
14
Q
What is G6PD?
A
The enzyme that catalyzes the initial step in the PPP, converts glucose-6-P to 6-phosphogluconolactone
- Commitment step
- Feedback inhibited by NADPH
15
Q
What is transketolase?
A
The enzyme that catalyzes the transfer of 2-carbon units among sugars in the PPP
16
Q
What is transaldolase?
A
The enzyme the catalyzes the transfer of 3-carbon units among sugars in the PPP
17
Q
What do transketolase and transaldolase do together?
A
Catalyze the reversible carbon shuffle reactions of the non-oxidative phase
18
Q
What are examples of the PPP in everyday biochemistry?
A
G6PD deficiency
- Most common enzyme deficiency in the world
- Affects >400 million people
- 90% decrease in enzyme activity results in inability of RBCs to make enough NADPH to protect the cells from ROS
- ROS generated by antimalarial drugs and compounds in fava beans
19
Q
Which reaction(s) in the oxidative phase of the PPP is/are reversible?
A
- Lactonase
20
Q
Which reaction(s) in the oxidative phase of the PPP is/are NOT reversible?
A
- G6PD
- 6PGD (6-Phosphogluconolactonate dehydrogenase)
21
Q
Since glucose-6-P is a substrate for glycolysis and the PPP, what controls the overall metabolic flux through these pathways?
A
The [NADP+]/[NADPH] in the cytosol
- Acts as a rheostat to regulate G6PD activity
22
Q
What is glutathione (GSH)?
A
Reduced form is an electron donor in redox reactions
23
Q
What are the electrons from GSH used for?
A
- To keep cysteine residues in hemoglobin in a reduced state
- To reduce ROS and hydroxyl free radicals that damage proteins and lipids via oxidation-induced cleavage reactions
24
Q
What is primaquine?
A
Antimalarial drug that causes oxidative stress in RBCs, which creates a hostile environment for the malaria parasite
- Leads to acute hemolytic anemia in people with a G6PD deficiency
25
What is gluconeogenesis?
Metabolic pathway used for the production of glucose from noncarbohydrate sources
26
How many enzymes do glycolysis and gluconeogenesis share?
7 enzymes
27
Which enzymes are glycolytic-specific?
- Hexokinase
- Phosphofructokinase-1
- Pyruvate kinase
28
What are the carbon sources for gluconeogenesis?
- Lactate
- Amino acids
- Glycerol
29
How do plants get carbon for gluconeogenesis?
Use the Calvin cycle to convert CO2 to glyceraldehyde-3-P
30
What does gluconeogenesis accomplish for the cell?
- Liver and kidneys use it to generate glucose from noncarbohydrate sources for export to other tissues that use glucose for energy
- Plants use it to convert CO2 to glyceraldehyde-3-P
31
What is the overall net reaction of gluconeogenesis?
2 Pyruvate + 2 NADH + 4 ATP + 2 GTP + 6 H2O --> 2 NAD+ + 2 H+ + 4 ADP + 2 GDP + 6 Pi
32
What are the key enzymes in gluconeogenesis?
- Pyruvate carboxylase
- Phosphoenolpyruvate carboxykinase
- FBPase-1
- Glucose-6-phosphotase
33
What is pyruvate carboxylase?
ATP-dependent enzyme that carboxylates **pyruvate to generate oxaloacetate**
- MITOCHONDRIAL ENZYME
34
What does pyruvate carboxylase maintain flux through?
Maintains flux through the citric acid cycle in the presence of acetyl-CoA
35
What is phosphoenolpyruvate carboxykinase?
Catalyzes a decarboxylation reaction in gluconeogenesis that converts **oxaloacetate to phosphoenolpyruvate and CO2**
36
What is FBPase-1?
Converts **fructose-1,6-BP to fructose-6-P** and opposes the PFK-1 in glycolysis
37
What is glucose-6-phosphotase?
Converts **glucose-6-P to glucose** and opposes the hexokinase/glucokinase reaction in glycolysis
38
What are examples of gluconeogenesis in everyday biochemistry?
Cori cycle
- Short intense exercise --> lactic acid build up in muscle because of anaerobic glycolysis
- Warm down period to increase circulation and remove lactate from muscle
- Lactate goes to liver where it's converted to glucose and sent back to muscle to replenish glycogen
39
What are the 3 bypass reactions in gluconeogenesis?
1. Pyruvate carboxylase and phosphoenolpyruvate carboxykinase (pyruvate kinase)
2. FBPase-1 (PFK-1)
3. Glucose-6-phosphotase (hexokinase)
40
What happens in the first bypass reaction in gluconeogenesis?
- Pyruvate carboxylase uses phosphoryl transfer energy in ATP to drive carboxylation reaction that converts **pyruvate to oxaloacetate**
- Oxaloacetate is decarboxylated by phosphoenolpyruvate carboxykinase and converted to **phosphoenolpyruvate**
41
What is biotin?
Cofactor for pyruvate carboxylase
- Functions as carrier of carboxyl groups in enzymatic reactions
42
What happens in the second bypass reaction in gluconeogenesis?
Fructose-1,6-BP is converted to fructose-6-P
43
What does a low energy charge lead to in terms of gluconeogenesis/glycolysis?
Low energy charge --> high AMP levels --> **activation of PFK-1** (increase flux through glycolysis) and **inhibition of FBPase-1** (decrease flux through gluconeogenesis)
44
What happens in the third bypass reaction in gluconeogenesis?
Free glucose is generated that can be exported to blood
45
Where is glucose-6-phosphotase located?
ER lumen
46
Where is hexokinase located?
Cytosol
47
What does fructose-2,6-BP do?
Reciprocally regulates glycolysis and gluconeogenesis
- Increase flux through glycolysis
- Decrease flux through gluconeogenesis
48
What inhibits pyruvate kinase?
ATP and alanine (decrease flux through glycolysis)
49
What stimulates pyruvate carboxylase?
Acetyl-CoA (increase flux through gluconeogenesis)
50
What is PFK-2/FBPase-2?
Dual-function enzyme that catalyzes the interconversion of fructose-6-P and fructose-1,6-BP
51
What are the 2 functional domains of PFK-2/FBPase-2?
1. **Kinase activity** that phosphorylates fructose-6-P into fructose-1,6-BP
2. **Phosphotase activity** that dephosphorylates fructose-1,6-BP into fructose-6-P
52
What is the Cori cycle?
Mechanism where lactate produced by anaerobic glycolysis in muscle is converted in liver cells to glucose via gluconeogenesis
53
Where do glycogen degradation and synthesis occur?
In the cytosol
54
What is the substrate for glycogen degradation and synthesis?
Nonreducing (free) ends
55
What kind of bonds does glycogen have?
α(1-->4) and α(1-->6)
56
What are the 4 key enzymes in glycogen degradation and synthesis?
1. Glycogen phosphorylase
2. Glycogen synthase
3. Glycogen branching enzyme
4. Glycogen debranching enzyme
57
What does glycogen phosphorylase do?
Catalyzes a phosphorolysis reaction using inorganic phosphate to **remove glucose from nonreducing ends of glycogen**
58
What reaction does glycogen phosphorylase catalyze?
Glucose-1-P --> glucose-6-P
59
What is glucose-6-P used for?
Glycolysis in muscles or gets dephosphorylated in liver
60
What is glycogen phosphorylase activity activated by?
Epinephrine and glucagon signaling
61
What does glycogen synthase do?
**Adds glucose residues to nonreducing ends** of glycogen using UDP-glucose as the substrate
62
What is glycogen synthase activated by?
Insulin signaling
63
What do glycogen branching and debranching enzymes do?
Mediate formation and cleavage of α(1-->6) glycosidic bonds
64
What tissues have a lot of glycogen?
Liver and skeletal muscle
65
What are glycogen particles?
Formed by 20-40 glycogen core complexes
66
What % by weight is liver glycogen?
~10% by weight
67
What is liver glycogen for?
Glucose source for export when there are limited dietary sources (between meals)
68
What % by weight is muscle glycogen?
~1-2% by weight
69
What is muscle glycogen for?
Used to generate glucose-6-P (chemical energy source in aerobic and anaerobic
glycolysis)
70
What purpose does glycogen degradation and synthesis serve in animals?
- Liver glycogen: short term energy source
- Muscle glycogen: glucose source during exercise
71
What is the net reaction of glycogen degradation at nonreducing ends?
Glycogenn units of glucose + Pi —> Glycogen n-1 units of glucose + Glucose-1-P
72
What is the net reaction of glycogen synthesis?
Glycogenn units of glucose + Glucose-1-P + ATP + H2O —> Glycogen n+1 units of glucose + ADP + 2Pi
73
What is an example of glycogen degradation and synthesis and everyday biochemistry?
Endurance athletes carbohydrate loading
- Short period of intense exercise followed by ingesting 10g/kg of carbs --> 2x muscle glycogen in 24 hours
74
What enzyme initiates glycogen degradation?
Glycogen phosphorylase
75
What is pyridoxal phosphate (PLP)?
Coenzyme for glycogen phosphorylase
- Acid-base catalyst that donates proton to Pi
76
How is PLP bound to glycogen phosphorylase?
Through a lysine residue
77
What does it mean when glycogen phosphorylase is processive enzyme?
Stays attached to glycogen substrate a cleaves α(1-->4) bonds and releases glucose-1-P until it gets too close to α(1-->6) branch point
78
Glycogen phosphorylase removes glucose until it is how many glucose away from the branch point?
4 glucose
79
What are the 2 steps of the debranching enzyme remodeling the substrate?
1. Debranching enzyme transfers 3 glucose units to nearest nonreducing end
2. Bifunctional debranching enzyme cleaves all α(1-->6) bond to release free glucose
80
How much glucose-1-P vs. glucose does complete glycogen degradation release?
~90% glucose-1-P and ~10% glucose
81
What are the 2 conformations of glycogen phosphorylase?
- R state (active)
- T state (inactive)
82
What reaction does phosphoglucomutase catalyze in glycogen degradation?
Glucose-1-P --> Glucose-6-P
83
How does unphosphorylated glycogen phosphorylase shift from T state to R state?
By binding AMP (allosteric effector)
84
What shifts unphposphorylated glycogen phosphorylase back to the R state?
Glucose-6-P (allosteric inhibitor; competes with AMP)
85
What enzyme initiates glycogen synthesis?
Phosphoglucomutase
86
What reaction does phosphoglucomutase catalyze in glycogen synthesis?
Glucose-6-P --> Glucose-1-P
87
What does glycogen synthase use as a substrate to catalyze the glycosyltransferase reaction?
UDP-glucose
88
What is glycogen synthase activity controlled by?
- Inhibited by phosphorylation (shift to T state)
- Activated by dephosphorylation (shift to R state)
89
What are the 2 catalytic activities of glycogenin?
1. **Glycosyltransferase activity**: uses UDP-glucose as donor to generate O-linked glycosidic bond between glucose to tyrosine residue
2. **Glycogen synthase activity**: extends glycogen chain to 8 glucose residues
90
What happens when glucose is added to a liver cell extract?
- Rapid decrease in glycogen phosphorylase activity
- Steady increase in glycogen synthase activity
91
What does low blood glucose lead to?
Glucagon signaling
92
What does high blood glucose lead to?
Insulin signaling
93
What does protein phosphotase 1 do?
Dephosphorylates specific protein targets
94
What stimulates protein phosphotase 1 activity?
Insuling signaling
95
What causes von Gierke disease?
Deficiency in liver **glucose-6-phosphotase**
96
What causes Hers disease?
Defect in liver **glycogen phosphorylase**
97
What causes Pompe disease?
Deficiency in lysosomal **α-1,4-glucosidase**
98
What causes McArdle disease?
Defect in muscle **glycogen phosphorylase**
99
What causes Cori disease?
Defect in liver muscle **glycogen debranching enzyme** that impacts its ability to fully degrade glycogen molecules
100
What causes Andersen disease?
Defect in liver **glycogen branching enzyme** that results in format of large linear glycogen molecules
Biochem 2 (CHEM 441B)
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