Chapter 16- Glycolysis and Gluconeogenesis Flashcards
1
Q
Glycolysis
A
A metabolic pathway- the sequence of reactions that metabolizes one molecule of glucose to create 2 molecules of pyruvate. At the same time, 2 net ATP are produced. This is an anerobic process (does not require oxygen) because it evolved before oxygen accumulated in the atmosphere
2
Q
How is pyruvate processed?
A
It can be processed anaerobically to lactate (lactic acid fermentation) or ethanol (alcoholic fermentation). Under aerobic conditions, pyruvate can be completely oxidized to CO2, generating much more ATP
3
Q
Lactic acid fermentation
A
Pyruvate is anaerobically processed to make lactate
4
Q
Alcoholic fermentation
A
Pyruvate is processed anaerobically to make ethanol
5
Q
Gluconeogenesis
A
The process by which metabolic products, like pyruvate and lactate, are salvaged to synthesize glucose. This is because glucose is considered a precious fuel to the body
6
Q
Alpha amylase
A
A pancreatic enzyme that digests starch and glycogen. They are complex carbohydrates that have to be converted to simple carbohydrates for absorption by the intestine and transport in the blood. It cleaves the alpha 1,4 bonds of starch and glycogen, but not the 1,6 bonds. The products of the reaction are di- and trisaccharides maltose and maltotriose
7
Q
Alpha-glucosidase (maltase)
A
An enzyme that digests maltotriose and any other oligosaccharides that escaped digestion by the amylase. It also cleaves maltose into 2 glucose molecules. It is located on the surface of the intestinal cells
8
Q
Alpha-dextrinase
A
Further digests the limit dextrin- the material from starch and glycogen that is not digestible because of the alpha 1,6 bonds
9
Q
Sucrase
A
An enzyme located on the surface of the intestinal cells. It degrades the sucrose contributed by vegetables to make fructose and glucose.
10
Q
Lactase
A
An enzyme that is responsible for degrading the milk sugar lactose into glucose and galactose. It is also found on the surface of intestinal cells. The monosaccharides are transported into the cells lining the intestine and then into the bloodstream
11
Q
Why is glucose important for the body?
A
Almost all organisms use glucose. In mammals, glucose is the only fuel that the brain uses under nonstarvation conditions. It is also the only fuel that red blood cells are able to use.
12
Q
Why is glucose used as a prominent fuel instead of another monosaccharide? (3)
A
- Glucose is one of several monosaccharides formed from formaldehyde under prebiotic conditions- it might have been available as a fuel source of primitive biochemical systems
- Glucose is the most stable hexose. All hydroxyl groups in the ring conformation are equatorial, contributing to its stability
- Glucose has a low tendency to nonenzymatically
glycosylate proteins because it tends to have a ring conformation. Open chain monosaccharides can rearrange proteins to form a more stable structure, which makes the proteins less functional
13
Q
Which cells is the glycolytic pathway found in?
A
Basically all cells- both prokaryotic and eukaryotic
14
Q
Cytoplasmic supramolecular complexes
A
In eukaryotic cells, glycolytic enzymes are organized in cytoplasmic supramolecular complexes. This strategy is efficient due to substrate channeling between active sites
and prevents the release of any toxic intermediates.
15
Q
Stage 1 of glycolysis
A
The trapping and preparation phase- no ATP is generated. Glucose is converted into fructose 1,6-bisphosphate through phosphorylation, isomerization, and then a second phosphorylation. This stage traps glucose in the cell and modifies it so that it can be cleaved into 2 phosphorylated 3-carbon compounds.
16
Q
Stage 2 of glycolysis
A
ATP is harvested (2 molecules) when the 3 carbon fragments from the first stage are oxidized to pyruvate
17
Q
How does glucose enter the cell?
A
It enters the cell through specific transport proteins and is phosphorylated by ATP to form glucose 6-phosphate. G6P has negatively charged phosphoryl groups, so it can’t pass through the membrane and is not a substrate for glucose transporters. The addition of the phosphoryl group facilitates the eventual metabolism of glucose to make 3 carbon molecules in stage 1
18
Q
Hexokinase
A
The enzyme that catalyzes the transfer of the phosphoryl group from ATP to the hydroxyl group on carbon 6 of glucose, when it enters the cell. It requires magnesium for activity, which forms a complex with ATP. The phosphorylation process marks the beginning of stage one of glycolysis
19
Q
Kinases
A
Enzymes that catalyze the transfer of a phosphoryl group from ATP to an acceptor. Phosphoryl transfer is a fundamental reaction in biochemistry
20
Q
Hexokinase induced fit
A
The binding of glucose causes a conformational change in hexokinase. The two lobes of hexokinase move toward each other when glucose is bound and close the hexokinase cleft. The bound glucose becomes surrounded by protein, except for the hydroxyl group of carbon 6, which will accept the phosphoryl group from ATP.
21
Q
Why are glucose induced structural changes significant? (2)
A
- The environment around the glucose becomes more polar, favoring reaction between the hydrophilic hydroxyl group of glucose and the terminal phosphoryl group of ATP
- The change allows the kinase to exclude water, keeping water away from the active site. This prevents undesired hydrolysis of ATP
22
Q
Isomerization of glucose 6-phosphate
A
Glucose 6-phosphate is isomerized to form fructose 6-phosphate, which is a conversion of an aldose into a ketose
23
Q
Phosphoglucose isomerase
A
Catalyzes the isomerization of glucose 6-phosphate to fructose 6-phosphate. The reaction takes multiple steps because both glucose and fructose exist in cyclic forms. The enzyme opens the 6 membered ring of glucose 6-phosphate, catalyzes the isomerization, then promotes the formation of the 5 membered ring of fructose 6-phosphate. This reaction is readily reversible
24
Q
What marks the completion of the first stage of glycolysis?
A
The formation of fructose 1,6-bisphosphate. All reactions in stage 1 work toward this goal
25
Bis- prefix
Means that two separate monophosphoryl groups are present. This is different from the di- prefix, which means that the phosphoryl groups are connected by an anhydride bond
26
Phosphofructokinase (PFK)
An enzyme that sets the pace of glycolysis. It catalyzes the second phosphorylation of stage 1 of glycolysis. One molecule of ATP is used to phosphorylate fructose 6-phosphate to fructose 1,6-bisphosphate. This reaction is irreversible to prevent the reformation of glucose 6-phosphate
27
Final reaction of stage 1 of glycolysis
Fructose 1,6 bisphosphate is cleaved into glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP)- 3 carbon molecules. This reaction is readily reversible
28
Aldolase
Catalyzes the formation of GAP and DHAP from fructose 1,6-bisphosphate
29
Triose phosphate isomerase
Interconverts GAP and DHAP, allowing the DHAP to be further metabolized. GAP is on the glycolysis pathway and can be processed to pyruvate to yield ATP, whereas DHAP cannot. DHAP is a 3 carbon fragment that can be used to generate ATP and would otherwise be lost, so TPI catalyzes a reversible isomerization reaction to switch between isomers
30
Structure of triose phosphate isomerase
Consists of a central core of 8 parallel beta strands surrounded by 8 alpha helices. This is a structural motif called an alpha-beta barrel
31
TPI deficiency
Triose phosphate isomerase is the only glycolytic enzyme
for which genetic deficiency in expression can be lethal. It is characterized by severe hemolytic anemia and neurodegeneration
32
TPI mechanism
TPI catalyzes the transfer of a hydrogen atom from carbon 1 to carbon 2, which is an intramolecular oxidation-reduction. This isomerization of a ketose into an aldose uses an enediol intermediate.
33
Enediol intermediate formation (3)
1. Glutamate 165 acts as a general base catalyst and
removes a proton from C-1 of the substrate to form the enediol intermediate.
2. Glutamate 165, now acting as a general acid catalyst, donates a proton to C-2, while histidine 95 removes a proton from C-1.
3. The product is formed, and glutamate 165 and histidine 95 return to their initial states.
34
2 noteworthy features of TPI
1. It is a powerful catalyst- it's ratio for the isomerization of glyceraldehyde 3-phosphate is close to the diffusion controlled limit.
2. TPI suppresses an undesired side reaction- the decomposition of the enediol intermediate into methyl glyoxal and orthophosphate
35
Why is TPI considered a kinetically perfect enzyme?
Its rate of catalysis is near the diffusion limit. This means that catalysis takes place every time that enzyme and substrate meet. The diffusion controlled encounter of substrate and enzyme is the rate limiting step in catalysis
36
Methyl glyoxal
The enediol intermediate in glycolysis can decompose into methyl glyoxal and orthophosphate. This reaction is faster than isomerization but useless, so TPI prevents it from occurring. Methyl glyoxal is a highly reactive compound that can modify the structure and function of biomolecules like proteins and DNA.
37
How does TPI prevent the enediol intermediate from decomposing?
TPI prevents the enediol from leaving the enzyme. The intermediate is trapped in the active site by a loop of 10 residues. The loop acts as a lid on the active site, shutting it when the enediol is present and reopening it when isomerization is completed.
38
Glyceraldehyde 3-phosphate dehydrogenase
The enzyme that catalyzes the conversion of glyceraldehyde 3-phosphate into 1,3- bisphosphoglycerate (1,3- BPG). NAD+ is reduced to NADH in this reaction. This is the first reaction in the second stage of glycolysis. During this reaction, aldehyde is oxidized to a carboxylic acid by NAD+. The carboxylic acid and the orthophosphate are joined to form the acyl-phosphate product
39
1,3- bisphosphoglycerate
An acyl phosphate which has a high phosphoryl transfer potential. This molecule is formed from glyceraldehyde 3-phosphate to begin the second stage of glycolysis
40
Formation of glyceraldehyde 1,3-bisphosphate (2 steps)
1. The highly exergonic oxidation of carbon 1 in GAP to an acid
2. The highly endergonic formation of glyceraldehyde 1, 3-bisphosphate from the acid
41
Why does the formation of glyceraldehyde 1,3-bisphosphate require an intermediate?
The first step of this reaction is highly exergonic, while the second step is highly endergonic. If the 2 reactions took place in succession, the second reaction would require a lot of activation energy. Therefore, the two processes are coupled the the aldehyde oxidation (step 1) drives the formation of the acyl phosphate. This is done through the formation of an intermediate formed by the aldehyde oxidation. It is linked to the enzyme by a thioester bond. The intermediate reacts with orthophosphate to form the high energy compound 1,3-bisphosphoglycerate
42
Thioester intermediate
Formed during the second stage of glycolysis, when 1,3-bisphosphoglycerate is formed from glyceraldehyde 3-phosphate. It is required to couple the aldehyde oxidation and acyl phosphate formation steps. The thioester intermediate preserves much of the free energy released in the oxidation reaction
43
Free-energy profiles for glyceraldehyde oxidation followed by acyl-phosphate formation
With no coupling between the two processes of the reaction, the second step requires a large activation barrier, making the reaction very slow. Once the thioester intermediate is used, the activation energy required decreases drastically
44
Reaction mechanism of glyceraldehyde 3-phosphate
dehydrogenase (4 steps)
1. GAP reacts with a cysteine residue to form a
hemithioacetal.
2. A thioester is formed by the transfer of a hydride to NAD+.
3. NADH is exchanged for NAD+. The charge on NAD+
facilitates the attack by the phosphate on the thioester.
4. Phosphate attacks the thioester, forming the product 1,3-BPG.
45
Phosphoglycerate kinase
Catalyzes the transfer of the phosphoryl group from the acyl phosphate of 1,3- bisphosphoglycerate to ADP. This creates one ATP molecule and 3-phosphoglycerate as the products. 1,3-BPG is an energy rich molecule with a greater phosphoryl-transfer potential than that of ATP, so it is used to power the synthesis of ATP using ADP
46
Substrate-level phosphorylation
Using 1,3-BPG and phosphoglycerate kinase to form ATP, as well as 3-phosphoglycerate. This is because 1,3-BPG acts as the phosphate donor and is a substrate with high phosphoryl-transfer potential
47
Phosphoglycerate mutase
Catalyzes the reaction where 3-Phosphoglycerate is converted into 2-phosphoglycerate. This is a rearrangement reaction where the position of the phosphoryl group shifts. The reaction catalyzed by the mutase involves a phosphorylated enzyme intermediate and the substrate passing through the 2,3-bisphosphorylated form.
48
Enolase
Catalyzes a dehydration reaction (one molecule of water is produced) to form an enol phosphate (PEP). The dehydration reaction elevates the transfer potential of the phosphoryl group. Converts 2-phosphoglycerate into phosphoenolpyruvate (PEP)- PEP has a high phosphoryl transfer potential, which is useful in the conversion to pyruvate
49
Mutase
An enzyme that catalyzes the intramolecular shift of a chemical group- includes phosphoglycerate mutase
50
Phosphoenolpyruvate
Phosphoenolpyruvate is a high phosphoryl-transfer
compound because the presence of the phosphate traps the compound in the unstable form. Once the phosphoryl group is donated to ATP, the enol undergoes a conversion into the more stable ketone (pyruvate). Once pyruvate is formed, glycolysis ends
51
Pyruvate kinase
An enzyme that catalyzes the irreversible transfer of a phosphoryl group from the phosphoenolpyruvate to ADP. This generates an ATP molecule and a pyruvate molecule.
52
What is the energy source for the formation of phosphoenolpyruvate?
When pyruvate is formed from phosphoenolpyruvate, an internal oxidation-reduction occurs. Carbon 3 takes electrons from carbon 2 in the conversion of 2-phosphoglycerate into pyruvate. Carbon oxidation powers the synthesis of a compound with high phosphoryl transfer potential (the phosphoenolpyruvate), which ultimately allows the synthesis of ATP
53
How many net ATP molecules are formed during glycolysis?
2. Stage 1 of glycolysis requires 2 ATP molecules, during the reactions catalyzed by phosphoglycerate kinase and pyruvate kinase, ADP is used to produced a total of 2 ATP molecules
54
During glycolysis, how many pyruvate are formed per glucose molecule?
2
55
Phosphoglycerate mutase mechanism (3 steps)
1. The enzyme requires catalytic amounts of 2,3-bisphosphate (2,3- BPG) to maintain an active site histidine residue in a phosphorylated form
2. The phosphoryl group is transferred to 3-phosphoglycerate to reform 2,3- BPG
3. The mutase converts 2,3-BPG into 2-phosphoglycerate. The mutase retains the phosphoryl group to regenerate the modified histidine
56
Net reaction in the transformation of glucose into pyruvate (4 reactants, 5 products)
One glucose, 2 phosphates, 2 ADP, and 2 NAD+ yields:
2 pyruvate, 2 ATP, 2 NADH, 2 H+, and 2 water molecules
57
Why does NAD+ need to be regenerated?
The activity of glyceraldehyde 3-phosphate dehydrogenase generates 1,3-BPG, but it also reduces NAD+ to NADH. There are limited amounts of NAD+ in the cell, so NAD+ must be regenerated for glycolysis to proceed. Therefore, the final process in the pathway is the regeneration of NAD+ through the metabolism of pyruvate
58
Where is NAD+ derived from?
From the vitamin niacin (B3)- this is a dietary requirement for humans
59
NAD+ can be regenerated by (3)
Further oxidation of pyruvate to CO2 or by the formation of ethanol or lactate from pyruvate.
60
3 possible fates of pyruvate
1. Fermentation
(two types)- takes place in the absence of oxygen
2. Metabolism in the citric acid cycle and electron transport chain- oxygen serves as the final electron acceptor
61
Fermentation
An ATP generating process where organic compounds act as both donors and acceptors of electrons- electrons are removed from one organic compound and passed to another organic compound. NADH drops off electrons with an organic molecule, like pyruvate, so NAD+ can be regenerated
62
Pyruvate decarboxylase
Catalyzes the decarboxylation of pyruvate in the reaction to form ethanol. The removal of the CO2 group converts pyruvate into an aldehyde (acetaldehyde), because a hydrogen replaces the CO2. This reaction and forms an intermediate in the reaction to form ethanol to and regenerate NAD+ later on in the reaction. Pyruvate decarboxylase requires the coenzyme thiamine pyrophosphate, which is derived from the vitamin thiamine (B1)
63
Ethanol formation from pyruvate mechanism (2)
1. Decarboxylation of pyruvate- uses pyruvate decarboxylase
2. Reduction of acetaldehyde to ethanol by NADH- uses alcohol dehydrogenase
This reaction regenerates NAD+
64
Alcohol dehydrogenase
Catalyzes the reduction of acetaldehyde to ethanol by NADH, in the reaction converting pyruvate to ethanol. Its active site contains a zinc ion that is coordinated to the sulfur atoms of two cysteine residues and a nitrogen atom of histidine. The zinc ion polarizes the carbonyl group of the substrate to favor the transfer of a hybrid from NADH
65
In which organisms is ethanol formed from pyruvate?
In yeast and several other microorganisms
66
Alcoholic fermentation
The type of fermentation where pyruvate is converted to ethanol by pyruvate decarboxylase and alcohol dehydrogenase. This reaction regenerates NAD+.
Glucose, 2 phosphate, 2 ADP, and 2 H+ yields 2 ethanol, 2 carbon dioxide, 2 ATP, and 2 waters
67
Maintaining redox balance
The NADH produced by the glyceraldehyde 3-phosphate dehydrogenase reaction must be reoxidized to NAD+ for the glycolytic pathway to continue. In alcoholic fermentation, alcohol dehydrogenase oxidizes NADH and generates ethanol. There is no net oxidation-reduction reaction in alcoholic fermentation
68
Lactic acid fermentation
A process that converts pyruvate to lactate. NADH transfers electrons directly to pyruvate. The C=O in pyruvate becomes a carbon bonded to H and OH groups (this is lactate). The NADH that was formed formed in the oxidation of glyceraldehyde 3-phosphate is consumed in the reduction of pyruvate since it loses its hydrogen. NAD+ is regenerated in this process, which sustains the continued process of glycolysis in anaerobic conditions
69
Lactate dehydrogenase
Catalyzes the reduction of pyruvate by NADH to form lactate. 1 glucose molecule is metabolized to produce 2 lactate molecules. Lactate is the ending point of the fermentation.
70
When does lactic acid fermentation occur in the human body?
Certain types of skeletal muscles in most animals can also function anaerobically for short periods. During intense bursts of exercise, the ATP needs rise faster than the ability of the body to provide oxygen to the muscle. The muscle can function anaerobically, until fatigue sets in. The fatigue is partially caused by lactate buildup. The normal pH of muscle fibers is 7, but it can get as low as 6.3 during exercise
71
Citric acid cycle and the electron transport chain
This is the third possible fate of pyruvate. Pyruvate is completely oxidized through pyruvate processing to generate acetyl CoA and the citric acid cycle (to oxidize the acetyl group)
72
Acetyl coenzyme A (acetyl CoA)
The entry point to the oxidative citric acid cycle and ETC pathway. It is formed inside the mitochondria by the oxidative decarboxylation of pyruvate
73
How does the aerobic pathway regenerate NAD+
The electrons are transferred to the final acceptor in
the electron transport chain (i.e., O2) via aerobic
metabolism. As the electrons are delivered from NADH
to the electron transport chain, NAD+ is restored.
74
Aerobic pathway mechanism
Pyruvate, NAD+, and CoA are the reactants. They yield acetyl CoA, carbon dioxide, NADH, and H+
75
Fermentation is a relatively inefficient pathway, why is it used?
Fermentation doesn't yield a lot of energy, but it's used because oxygen is not required. This is beneficial for organisms living in soils, deep water, and skin pores, because it means they don't need oxygen to survive
76
Obligate anaerobes
Organisms that can't survive in the presence of oxygen, which is a highly reactive compound. The bacterium that causes tetanus is an example
77
Facultative anaerobes
Organisms that metabolize glucose aerobically when oxygen is present and perform fermentation when oxygen is absent
78
Metabolism of fructose
There are no catabolic pathways for metabolizing fructose, so the sugar is converted into a metabolite of glucose. Fructose metabolism mainly occurs in the liver, through the fructose 1-phosphate pathway
79
Fructokinase
The enzymes that catalyzes the phosphorylation of fructose to fructose 1-phosphate
80
Fructose 1-phosphate pathway
1. Fructose is converted to fructose 1-phosphate by fructokinase
2. Fructose 1-phosphate splits into glyceraldehyde and dihydroxyacetone phosphate (an intermediate in glycolysis)
3. Glyceraldehyde is phosphorylated to glyceraldehyde 3-phosphate
81
Fructose 1-phosphate aldolase
Catalyzes the split of fructose 1-phosphate into glyceraldehyde and dihydroxyacetone phosphate
82
Triose kinase
Catalyzes the phosphorylation of glyceraldehyde into glyceraldehyde 3-phosphate, a glycolytic intermediate
83
Hexokinase and fructose
Can phosphorylate fructose to fructose 6-phosphate in tissues like adipose tissue (instead of the liver)
84
Fructose and galactose are converted to
Fructose, from table sugar or high fructose corn syrup,
and galactose, from milk sugar, can be converted into
glycolytic intermediates
85
Entry Points in Glycolysis for
Fructose and Galactose
Galactose enters as glucose 6-phosphate. In adipose tissue, fructose can enter as fructose 6-phosphate. In the liver, fructose can enter as DHAP or GAP
86
Consequences of excess fructose consumption
Fructose is a commonly used sweetener. Excess
consumption of fructose has been linked to fatty liver,
insulin insensitivity, obesity, and type 2 diabetes.
87
Why does excess consumption of fructose cause health problems?
These disorders are the result of how fructose is processed by the liver. The actions of fructokinase and triose kinase bypass the important regulatory step in glycolysis, which is the phosphofructokinase catalyzed reaction. Fructose-derived glyceraldehyde 3-phosphate and DHAP are processed by glycolysis to pyruvate and then to acetyl CoA in an unregulated fashion. Therefore, this excess acetyl CoA is converted to fatty acids, which can be transported to adipose tissue can cause obesity. The liver can also accumulate fatty acids, causing fatty liver. The activity of fructokinase and triose kinase can deplete the liver of ATP and inorganic phosphate, compromising liver function
88
Galactose-glucose interconversion pathway
Galactose is converted into glucose 6-phosphate
1. Galactose is converted into galactose 1-phosphate
2. G1P acquires a uridyl group from uridine diphosphate glucose (UDP glucose) which is an activated intermediate in the synthesis of carbohydrates- creates UDP-galactose and glucose 1-phosphate
3. The galactose component of UDP galactose is epimerized to glucose
4. Glucose 1-phosphate, formed from galactose, is isomerized to glucose 6-phosphate
89
Galactokinase
Catalyzes the phosphorylation of galactose to galactose 1-phosphate
90
Galactose 1-phosphate uridyl transferase
Catalyzes the reaction where galactose 1-phosphate acquires a uridyl group from UDP-glucose. The products of this reaction are UDP-galactose and glucose 1-phosphate
91
UDP-galactose 4-epimerase
Inverts the configuration of the hydroxyl group at carbon 4 of UDP-galactose
92
Phosphoglucomutase
Isomerizes glucose 1-phosphate to glucose 6-phosphate
93
Sum of the reactions in the galactose-glucose interconversion pathway
Galactose and ATP yield glucose 1-phosphate, ADP, and H+
94
Why is the conversion of UDP-glucose into UDP-galactose important?
It is essential for the synthesis of galactosyl residues in complex polysaccharides and glycoproteins if the amount of galactose in the diet is inadequate to meet these needs
95
Lactose intolerance (hypolactasia)
Gastrointestinal issues when consuming milk, caused by a deficiency of the enzyme lactase, which cleaves lactose into glucose and galactose. A decrease in lactase is normal in the course of development in all mammals. However, this decrease with weaning from breast milk is not as pronounced in certain groups, especially Northern Europeans. This is caused by a mutation that probably was selected for because people that were able to consume milk in certain geographic regions had a survival advantage.
96
What happens to the lactose in the intestine of a lactase-deficient person?
The lactose is a good energy source for microorganisms in the colon, and they ferment it to lactic acid while generating methane (CH4) and hydrogen gas (H2). The gas produced creates an uncomfortable feeling of gut distension. The lactate produced by the microorganisms is osmotically active and draws water into the intestine, as does any undigested lactose, causing diarrhea. The anaerobic lactobacillus bacteria is one example of bacteria that ferments glucose into lactic acid. It is used in food and is a normal component of the microbiome
97
Classic galactosemia
The most common form of disruption of galactose metabolism. It is an inherited deficiency in galactose 1-phosphate uridyl transferase activity. Symptoms include failure to thrive, jaundice, and liver enlargement that can lead to cirrhosis. Cataract formation may also occur. The most common treatment is to remove galactose (and lactose) from the diet .
98
Cataract formation
Clouding of the normally clear lens of the eye. If the transferase is not active in the lens of the eye, the presence of aldose reductase causes the accumulating galactose to be reduced to galactitol. Galactitol is poorly metabolized and accumulates into the lens. Water will diffuse into the lens to maintain osmotic balance, triggering the formation of cataracts
99
Enzymes catalyzing irreversible reactions in glycolysis (3)
1. Hexokinase
2. Phosphofructokinase
3. Pyruvate kinase
These enzymes are potential control sites to regulate the pathway
100
Regulation of muscle glycolysis
Muscle glycolysis is primarily controlled by the energy charge of the cell- the ratio of ATP to AMP. A high concentration of ATP inhibits PFK, pyruvate kinase, and hexokinase. Glucose 6-phosphate is converted into glycogen at this time. During exercise, muscle contraction causes a decrease in the ATP/AMP ratio. This activates PFK and hence glycolysis
101
How does phosphofructokinase regulate glycolysis?
PFK is the most important control site. High levels of ATP allosterically inhibit the enzyme. ATP binds to a specific regulatory site that is distinct from the catalytic site. The binding of ATP lowers the enzyme's affinity for fructose 6-phosphate. AMP reverses the inhibitory action of ATP, so the activity of the enzyme increases when the ATP/AMP ratio is lowered (the energy charge falls). A decrease in PH also inhibits PFK activity by augmenting the inhibitory effect of ATP- this might occur when a muscle produces too much lactic acid. The inhibitory effect protects the muscle from damage that would result from the accumulation of too much acid
102
Allosteric regulation of phosphofructokinase
A high level of ATP inhibits the enzyme by decreasing its affinity for fructose 6-phosphate. Therefore, when levels of ATP are high, reaction velocity decreases
103
Why is AMP, not ADP, the positive regulator of
phosphofructokinase?
AMP has become the signal for the low energy state due to the action of adenylate kinase. Also, the use of AMP as an allosteric regulator provides a sensitive control, because in the cell, ATP is greater in concentration than ADP, and ADP is greater in concentration than AMP, so small fluctuations in ATP concentration are magnified into large changes in AMP concentration. The magnification of small changes in ATP to larger changes in AMP leads to tighter control by increasing the range of sensitivity of phosphofructokinase
104
Adenylate kinase
When ATP is being utilized rapidly and ATP needs are great, adenylate kinase can form ATP from ADP. 2 ADP molecules are used to produce ATP and AMP. Therefore, some ATP is salvaged from ADP, and AMP becomes the signal for the low energy state (since adenylate kinase is only used when ATP is low)
105
Regulation of hexokinase
Hexokinase is inhibited by its product, glucose 6-phosphate. High concentrations of this molecule signal that the cell no longer requires glucose for energy or for the synthesis of glycogen (the storage form of glucose), and glucose will be left in the blood. Glucose 6-phosphate concentration is a means by which PFK communicates with hexokinase
106
Why is PFK rather than hexokinase the pacemaker of glycolysis?
Glucose 6-phosphate is not solely a glycolytic intermediate. In muscle, glucose 6-phosphate can also be converted into glycogen. The first irreversible reaction (committed step) is unique to the glycolytic pathway. Therefore it makes sense that PFK is the primary control site in glycolysis. The enzyme catalyzing the committed step is the most important control element in the pathway
106
Regulation of pyruvate kinase
Pyruvate kinase is allosterically inhibited by ATP, to slow glycolysis when the energy charge is high. When the pace of glycolysis increases, fructose 1,6-bisphosphate activates the kinase to keep pace with the oncoming influx of intermediates. This process is called feedforward stimulation
107
How is phosphofructokinase inhibited in the liver?
PFK is inhibited by citrate, which is an early intermediate in the citric acid cycle. A high level of citrate means that precursors of biomolecules are abundant, so there's no need to degrade more glucose for this purpose. Citrate inhibits PFK by enhancing the inhibitory effect of ATP
108
How is phosphofructokinase activated in the liver?
It is activated by fructose 2,6-bisphosphate. The concentration of fructose 6-phosphate rises when blood glucose concentration is high- the abundance of fructose 6-phosphate accelerates the synthesis of F26B, which acts as an intermediate. Therefore, an abundance of fructose 6-phosphate leads to a higher concentration of F26B. The binding of F26B increases the affinity of PFK for fructose 6-phosphate and diminishes the inhibitory effect of ATP. Glycolysis accelerates when glucose is abundant
109
Glucokinase
An enzyme in the liver that is a specialized isozyme of hexokinase. Glucokinase is not inhibited by glucose 6-phopshate. It provides glucose 6-phosphate for the synthesis of glycogen and for the formation of fatty acids.
110
When is glucokinase active?
Glucokinase is active only after a meal, when blood-glucose levels are high. It phosphorylates glucose only when glucose is abundant because the affinity of glucokinase for glucose is around 50 times lower than that of hexokinase. Also, when glucose concentration is low, glucokinase is inhibited by the liver-specific glucokinase regulatory protein (GKRP), which sequesters the kinase in the nucleus until the glucose concentration increases
111
Importance of glucokinase's low affinity for glucose
The low affinity for glucose gives the brain and muscles first call on glucose when its supply is limited, and ensures that glucose will not be wasted when it is abundant
112
Isozymic forms of pyruvate kinase (2)
1. L type predominates in the liver
2. M type predominates in muscle and the brain
113
L type vs M type of pyruvate kinase
The liver and muscle enzymes mostly behave the same in regards to allosteric regulation, but the liver enzyme is also inhibited by alanine, which is a signal that building blocks are available. The 2 forms also differ in their susceptibility to covalent modification. The catalytic properties of the L form (not M) are also controlled by reversible phosphorylation
114
Reversible phosphorylation
Regulates the catalytic activity of the L form of pyruvate kinase in the liver. When the blood glucose level is low, the glucagon triggered cyclic AMP cascade triggers the synthesis of glucagon and leads to the phosphorylation of pyruvate kinase, which diminishes the activity of pyruvate kinase. This phosphorylation is hormone triggered and prevents the liver from consuming glucose when it's more urgently needed by the brain and muscle. Once blood glucose concentration is adequate, the enzyme is dephosphorylated and activated
115
Glucose transporters
There are 5 glucose transporters (GLUT1-GLUT5), which all consist of a single polypeptide chain that is around 500 residues long. Each glucose transporter has a 12 transmembrane helix structure. They are responsible for mediating the downhill movement of glucose across the plasma membrane of animal cells
116
GLUT1 and GLUT3
Present in nearly all mammalian cells and are responsible for basal glucose uptake. They continually transport glucose into cells at an essentially constant rate
117
GLUT2
Present in the liver and pancreatic beta cells. It has a very high KM value for glucose, meaning that glucose enters these tissues at a biologically significant rate only when there is a lot of glucose in the blood. The pancreas can sense the glucose level and accordingly adjust the rate of insulin secretion.
118
GLUT4
Transports glucose into muscle and fat cells. The number of GLUT4 transporters in the plasma membrane increases rapidly in the presence of insulin, which signals the fed state. Therefore, insulin promotes the uptake of glucose by muscle and fat. Endurance training increases the amount of this transporter present in muscle membranes
119
GLUT5
Present in the small intestine, functions primarily as a fructose transporter
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Aerobic glycolysis (Warburg effect)
Tumors display enhanced rates of glucose uptake and glycolysis. Rapidly growing tumor cells will metabolize glucose to lactate even in the presence of oxygen. This occurs even in leukemias, which do not form tumors
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Selective advantage of aerobic glycolysis (4)
1. Aerobic glycolysis secretes lactic acid that is then secreted. Acidification of the tumor environment has been shown to facilitate tumor invasion.
2. Lactate also impairs the activation of CD8, T, and NK immune system cells that would normally attack the tumor.
3. The increased uptake of glucose and formation of glucose 6-phosphate provides substrates for the pentose phosphate pathway, which generates NADPH (biosynthetic reducing power)
4. Cancer cells grow more rapidly than the blood vessels that nourish them. Solid tumors begin to experience hypoxia as they grow. The use of aerobic glycolysis reduces the dependence of cell growth on oxygen
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What biochemical alterations facilitate the switch to aerobic glycolysis?
Changes in gene expression of isozymic forms of 2 glycolytic enzymes may occur. Tumor cells express an isozyme of hexokinase that binds to mitochondria, so the enzyme has access to any ATP generated by oxidative phosphorylation and is not susceptible to feedback inhibition by its product, glucose 6-phosphate. Pyruvate kinase M is also expressed, wich has a lower catalytic rate than normal pyruvate kinase. This allows the use of glycolytic intermediates for biosynthetic processes required for cell proliferation.
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How are tumors visualized
Using 2-18F-2-D-deoxyglucose (FDG) and positron emission tomography. When patients are infused with a non-metabolizable analog of glucose, tumors are readily visualized by tomography. This is because tumors demonstrate increased metabolism
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Hypoxia-inducible transcription factor (HIF-1)
A transcription factor activated by the hypoxia that some tumors experience with rapid growth. It increases the expression of most glycolytic enzymes and the glucose transporters GLUT1 and GLUT3. These adaptations help the tumor to survive until blood vessels can grow. It also increases the expression of signal molecules like vascular endothelial growth factor, that facilitates the growth of blood vessels that provide nutrients to the cells. Without blood vessels, the tumor would cease to grow and either die or remain very small
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How is anaerobic exercise training affect glycolysis?
Anaerobic exercise training forces the muscles to rely on lactic acid fermentation for ATP production. Similar to cancer, HIF-1 is activated. It produces the same effects as those seen in the tumor- enhanced ability to generate ATP anaerobically and a stimulation of blood vessel growth. These biochemical effects account for the improved athletic performance that results from training and demonstrates how behavior can affect biochemistry
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Proteins in glucose metabolism encoded by genes regulated by hypoxia-inducible factor (9)
1. GLUT1
2. GLUT3
3. Hexokinase
4. Phosphofructokinase
5. Aldolase
6. Glyceraldehyde 3-phosphate dehydrogenase
7. Phosphoglycerate kinase
8. Enolase
9. Pyruvate kinase
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Alteration of gene expression in tumors due to hypoxia
The hypoxic conditions in a tumor lead to the activation of HIF-1, which induces metabolic adaptation (an increase in glycolytic enzymes) and activates angiogenic factors that stimulate the growth of new blood vessels
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Gluconeogenesis
The synthesis of glucose from noncarbohydrate precursors. Pyruvate is converted into glucose. Maintaining levels of glucose is important because the brain depends on glucose as its primary fuel and RBCs use glucose as their only fuel. Gluconeogenesis is important during a longer period of fasting or starvation, since the body's glucose reserves are only sufficient to meet glucose needs for around a day
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The major precursors for gluconeogenesis (3)
lactate, amino acids, and glycerol
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Major site of gluconeogenesis
The major site of gluconeogenesis is the liver, although some gluconeogenesis can occur in the kidney
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Entry points for glycerol into the gluconeogenic cycle
Glycerol enters at DHAP, during the TPI reaction
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Why isn't gluconeogenesis considered a reversal of glycolysis?
Several reactions must differ because the equilibrium of glycolysis lies far on the side of pyruvate formation. Most of the decrease in free energy in glycolysis takes place in 3 irreversible steps- the reactions catalyzes by hexokinase, PFK, and pyruvate kinase. However, in gluconeogenesis, these irreversible reactions of glycolysis must be bypassed
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Pyruvate carboxylase
Catalyzes the first step in gluconeogenesis- the carboxylation of pyruvate to form oxaloacetate at the expense of an ATP molecule. This reaction occurs in the mitochondria. It requires the vitamin biotin as a cofactor
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Biotin
A covalently attached prosthetic group- it is attached to pyruvate carboxylase. It serves as a carrier of activated carbon dioxide. The carboxylase group of biotin is linked to the E-amino group of a lysine residue by an amide bond
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Pyruvate carboxylase reaction mechanism
Uses pyruvate, carbon dioxide, ATP, and water, to yield oxaloacetate, ADP, phosphate, and 2 H+
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3 stages of a pyruvate carboxylase reaction
1. The biotin carboxylase domain catalyzes the formation carboxyphosphate.
2. The carboxylase then transfers the CO2 to the biotin carboxyl carrier protein (BCCP).
3. The BCCP carries the activated CO2 to the pyruvate
carboxylase domain, where the CO2 is transferred to pyruvate
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Pyruvate carboxylase structure
It's a tetramer composed of 4 identical subunits, and each subunit consists of 4 domains. BC (the first domain) catalyzes the formation of carboxyphosphate and the subsequent attachment of carbon dioxide to BCCP (the second domain). BCCP is the site of covalently attached biotin. Once bound to carbon dioxide, VCCP leaves the biotin carboxylase active site and swings almost the entire length of the subunit to the active site of the carboxyl transferase domain (the third domain), which transfers the CO2 to pyruvate to form oxaloacetate
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Function of the 4th domain of pyruvate carboxylase
The fourth domain (PT) facilitates the formation of the tetramer and is the binding site for acetyl CoA, which is a required allosteric activator for carboxylation of biotin. How acetyl CoA facilitates the carboxylase reaction is unclear
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Conversion of oxaloacetate into phosphoenolpyruvate (3)
1. Oxaloacetate has to be transported to the cytoplasm to complete PEP synthesis. Oxaloacetate is reduced to malate
2. Malate is transported across the mitochondrial membrane and reoxidized to oxaloacetate
3. Oxaloacetate is simultaneously decarboxylated and phosphorylated to generate phosphoenolpyruvate
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Malate dehydrogenase
Reduces oxaloacetate to malate so oxaloacetate can be transported to the cytoplasm to complete PEP synthesis
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NAD+ linked malate dehydrogenase
Reoxidizes malate to oxaloacetate after it crosses the mitochondrial membrane. This reaction also provides NADH for use in subsequent steps in gluconeogenesis
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Phosphoenolpyruvate carboxykinase (PEPCK)
Simultaneously decarboxylates and phosphorylates oxaloacetate to generate phosphoenolpyruvate. The phosphoryl donor is GTP. The carbon dioxide that was added to pyruvate by pyruvate carboxylase comes off in this step
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Compartmental cooperation in gluconeogenesis
The first reactions of gluconeogenesis occur in the mitochondria. Carboxylation of pyruvate to form oxaloacetate occurs in the mitochondrial matrix. Then, oxaloacetate leaves the mitochondrion by a specific transport system in the form of malate, which is reoxidized to form oxaloacetate in the cytoplasm
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Sum of the reactions catalyzed by pyruvate
carboxylase and phosphoenolpyruvate carboxylase
Pyruvate, ATP, GTP, and water yields PEP, ADP, GDP, phosphate and 2 H+
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Fructose 1,6-bisphosphatase
Once PEP is formed, it's metabolized by enzymes of glycolysis in the reverse direction- the reverse reactions take place until the next irreversible step is reached. This irreversible step is the hydrolysis of fructose 1,6 bisphosphate to fructose 6-phosphate and phosphate. Fructose 1,6-bisphosphatase is responsible for catalyzing this reaction
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Final step in the generation of free glucose
Fructose 6-phosphate that was generated by fructose 1,6-bisphosphatase is converted into glucose 6-phosphate. The final step in the reaction takes place in the liver. Glucose 6-phosphate is transported into the lumen of the endoplasmic reticulum, and is hydrolyzed to glucose by glucose 6-phosphatase
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Glucose 6-phosphatase
An integral membrane protein on the inner surface of the endoplasmic reticulum, catalyzes the formation of glucose from glucose 6-phosphate in the liver. Glucose and phosphate are then shuttled back to the cytoplasm by a pair of transporters
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When does gluconeogenesis end?
In most tissues, gluconeogenesis ends with the formation of glucose 6-phosphate. Free glucose isn't generated because most tissues lack glucose 6-phosphatase. In this situation, glucose 6-phosphate is converted into glycogen, which is the storage form of glucose
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Why do gluconeogenesis and glycolysis need to be reciprocally regulated?
Gluconeogenesis and glycolysis are regulated so that within a cell, one pathway is relatively inactive whereas the other is highly active. The rationale for reciprocal regulation is that glycolysis will predominate when glucose is abundant and gluconeogenesis will be highly active when glucose is scarce.
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What determines whether glycolysis or gluconeogenesis will be most active?
Energy charge- if ATP is required, glycolysis predominates. If glucose is required, gluconeogenesis is favored. The interconversion of fructose 6-phosphate and fructose 1,6-bisphosphate is the first important regulatory point. At this point, the concentration of AMP is high. AMP stimulates PFK and inhibits fructose 1,6-bisphosphatase, which turns on glycolysis. Another regulatory point is that the interconversion of phosphoenolpyruvate and pyruvate in liver. The glycolytic enzyme pyruvate kinase is inhibited by ATP and alanine, which signals that the energy charge is high and that building blocks are abundant
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Regulatory role of fructose 2,6-bisphosphate
In liver, the rates of glycolysis and gluconeogenesis are
adjusted to maintain blood-glucose levels. The key regulator of glucose metabolism in liver is fructose
2,6-bisphosphate. Fructose 2,6-bisphosphate stimulates
phosphofructokinase and inhibits fructose 1,6 bisphosphatase. When blood glucose is low, fructose 2,6-bisphosphate loses a phosphoryl group to form fructose 6-phosphate
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How is the concentration of fructose 2,6-bisphosphate controlled to rise and fall with blood glucose levels?
Two enzymes regulate its concentration. One phosphorylates fructose 6-phosphate and the other dephosphorylates fructose 2,6-bisphosphate.
1. Fructose 2,6-bisphosphate is formed in a reaction catalyzed by phosphofructokinase 2 (PFK2).
2. Fructose 6-phosphate is formed through the hydrolysis of fructose 2,6-bisphosphate by a specific phosphatase, fructose bisphosphatase 2 (FBPase2)
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Bifunctional enzyme
The kinase (PFK2) that synthesizes fructose 2,6-bisphosphate and the phosphatase (FBPase2) that hydrolyzes this molecule are located on the same polypeptide chain. The enzyme contains a kinase domain that is fused to a phosphatase domain. Phosphorylation of the bifunctional enzyme activates the phosphatase activity and inhibits the kinase activity.
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Control of the synthesis and degradation of fructose 2,6-bisphosphate
A low blood glucose concentration is signaled by glucagon and leads to the phosphorylation of the bifunctional enzyme. The activities of PFK2 and FBPase2 are reciprocally controlled by the phosphorylation of a single serine residue. This phosphorylation of the enzyme and therefore to a lower concentration of fructose 2,6-bisphosphate- this slows glycolysis. Insulin accelerates the formation of fructose 2,6-bisphosphate by facilitating the dephosphorylation of the bifunctional enzyme
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Substrate cycle
A pair of reactions such as the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate and its hydrolysis back to fructose 6-phosphate. Both reactions are not fully active at the same time because of reciprocal allosteric controls
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Futile cycle
It has now been shown though that there can be some detectable activity of opposing pathways at the same time. Previously this was considered undesirable and was termed a futile cycle. However, it is now believed that substrate cycles can sometimes be biologically important, enhancing metabolic signals. A small change in the rates of the two opposing reactions can result in a large change in the net flux.
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Purpose of lactate in skeletal muscles
In contracting fast twitch skeletal muscle fibers during vigorous exercise, the rate at which glycolysis produces pyruvate exceeds the rate at which the citric acid cycle oxidizes it. In this case, lactate dehydrogenase reduces excess pyruvate to lactate to restore redox balance. In contracting skeletal muscle, the formation and release of lactate lets the muscle generate ATP in the absence of oxygen and shifts the burden of metabolizing lactate and shifts the burden of metabolizing lactate to other organs
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Why does lactate need to be released from the muscle?
Lactate is a dead end in metabolism. It has to be converted back to pyruvate before it can be metabolized. Both pyruvate and lactate diffuse out of these cells through carriers into the blood. Its release from the muscle lets the muscle generate ATP in the absence of oxygen and shifts the burden of metabolizing lactate and shifts the burden of metabolizing lactate to other organs
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2 fates of pyruvate and lactate in the bloodstream
1. The plasma membranes of some cells, especially cells in cardiac muscle and slow twitch skeletal muscle, contain carriers that make the cells highly permeable to lactate and pyruvate.
2. Excess lactate enters the liver and is converted first into pyruvate and then into glucose by the gluconeogenic pathway
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Cell permeability to pyruvate and lactate
Cells in cardiac muscle and slow twitch skeletal muscle contain carriers in their plasma membranes that make the cells permeable to pyruvate and lactate. These molecules diffuse from the blood into permeable cells. Inside the well oxygenated cells, lactate can be reverted back to pyruvate and metabolized through the citric acid cycle and oxidative phosphorylation to generate ATP. The use of lactate instead of glucose makes more circulating glucose available to active muscle cells
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How does the liver use lactate?
Lactate enters the liver and is converted first into pyruvate and then into glucose through gluconeogenesis. Contracting skeletal muscle supplies lactate to the liver, which uses it to synthesize and release glucose. Therefore, the liver restores the level of glucose necessary for active muscle cells, which derive ATP from the glycolytic conversion of glucose into lactate (Cori cycle)
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Cori cycle
Lactate formed by active muscle is converted into glucose by the liver. The liver restores the level of glucose necessary for active muscles, which derive ATP from the glycolytic conversion of glucose into lactate. This cycle shifts part of the metabolic burden of active muscle to the liver
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Role of alanine
Alanine, like lactate, is a major precursor of glucose in the liver. Alanine is generated in muscle when the carbon skeletons of some amino acids are used as fuels. The nitrogens from these amino acids are transferred to pyruvate to form alanine- the reverse reaction takes place in the liver
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Cooperation between glycolysis and gluconeogenesis
Glycolysis and gluconeogenesis are coordinated, in a tissue specific fashion, to ensure the energy needs of all cells are met. In skeletal muscle, glucose will be metabolized aerobically to carbon dioxide and water, or it can be metabolized anaerobically to lactate. In cardiac muscle, the lactate can be converted into pyruvate and used as a fuel, along with glucose, to power the heartbeat. Gluconeogenesis takes place in the liver so there is enough glucose present in the blood for skeletal and cardiac muscle, as well as other tissues
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How are glycolysis and gluconeogenesis evolutionarily intertwined?
The second part of glycolysis, the metabolism of trioses, is common to both glycolysis and gluconeogenesis. The four enzymes catalyzing the metabolism of these trioses are present in all species. In contrast, the enzymes of the first part of glycolysis, the metabolism of hexoses, are not nearly as conserved. The common part of the two pathways may be the oldest part, to which other reactions were added during the course of evolution
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Triose Phosphate Isomerase Deficiency (TPID)
TPID is a multisystem disorder that presents in early childhood. The symptoms include congenital red blood cell defects and progressive neuromuscular disorder, including inflammation and damage of the heart muscle. Death in early childhood may result. Dihydroxyacetone phosphate accumulates in cells, especially red blood cells.
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Why is TPID so serious?
The central nervous system and red blood cells rely completely on glucose metabolism for energy, which is why they are dramatically impacted when this enzyme is absent. Because of the enzyme’s position in the glycolytic pathway, three of the six carbons derived from glucose cannot be used if this enzyme is not functional, which would impact ATP production. In addition, it is believed that DHAP buildup would lead to its conversion to the toxic intermediate methylglyoxal, which is highly reactive and can bind to proteins and lead to advanced glycation end products (AGE), which inhibit protein function
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Pyruvate Carboxylase Deficiency (PCD)
Two of the primary features in this case are hypoglycemia and lactic acidosis. Hypoglycemia results from the inability to perform gluconeogenesis due to the missing pyruvate carboxylase. Liver also normally removes lactic acid from the blood and uses it as a gluconeogenic precursor. However, if this is unable to proceed because of a block at the pyruvate carboxylase step, then lactic acid will remain in the blood, leading to a drop in blood pH (acidosis).
