Chapter 16- Glycolysis and gluconeogenesis Flashcards
Glycolysis
Glycolysis breaks down one molecule of glucose into two molecules of pyruvate, and 2 molecules of ATP are produced as a part of this process. This process is anaerobic- it does not require oxygen. This is because it evolved before substantial amounts of oxygen accumulated in the atmosphere
After pyruvate is produced during glycolysis, what are the ways it can be further processed? (3)
- Anaerobically, lactate is formed through lactic acid fermentation
- Anaerobically, ethanol is formed through alcoholic fermentation
- Under aerobic conditions, pyruvate can be completely oxidized to form carbon dioxide and generate much more ATP.
During glycolysis, the fates of pyruvate depend on
The organism and whether or not oxygen is present.
Gluconeogenesis
Gluconeogenesis is a process that salvages metabolic products like pyruvate and lactate in order to synthesize glucose, since it’s such an important fuel to the body. Basically, gluconeogenesis uses the products of glycolysis to synthesize glucose.
How are glycolysis and gluconeogenesis different?
Although the two pathways have some enzymes in common, they are not just the reverse of each other. There are steps of glycolysis that are irreversible and release a high amount of energy. These steps are skipped in gluconeogenesis. In addition, the pathways are reciprocally regulated. This is so the pathways will not take place simultaneously in the cell.
Where is glucose generated from?
Glucose is generated from dietary carbohydrates. We generally consume starch and glycogen as part of our diets. These are complex carbohydrates that are converted to simple carbohydrates so they can be absorbed by the intestine and transported in the blood.
ɑ-amylase function
ɑ-amylase is a pancreatic enzyme that digests starch and glycogen
How does ɑ-amylase work, and which molecules does it result in?
Amylase cleaves (breaks) the ɑ-1,4 bonds of starch and glycogen, but not the ɑ-1,6 bonds. This results in di- and trisaccharides maltose and maltotriose.
Maltase
An enzyme that can digest maltotriose and all other oligosaccharides that were not digested by amylase. It also breaks the bonds of maltose, creating 2 glucose molecules.
Limit dextrin
The material that is not digested by amylase due to its ɑ-1,6 bonds is called the limit dextrin. ɑ-dextrinase is an enzyme that further digests the limit dextrin.
Enzymes found on the surface of intestinal cells (3)
ɑ-glucosidase, sucrase, and lactase
Sucrase
Enzyme that degrades the sucrose contributed by vegetables to fructose and glucose.
Lactase
Degrades the lactose that comes from milk into glucose and galactose.
What happens to the monosaccharides that result from enzyme reactions?
These monosaccharides contributed by enzyme reactions are transported into the cells lining the intestine and then into the bloodstream.
Which animals use glucose as fuel?
Almost all animals use glucose. In mammals, glucose is the only fuel that the brain uses under non starvation conditions and the only fuel that red blood cells can use at all.
Why is glucose used as a prominent fuel, opposed to another monosaccharide? (3)
- Glucose is one of several monosaccharides formed from formaldehyde under prebiotic conditions. It might have been used for primitive biochemical systems.
- Glucose has a low tendency to nonezymatically glycosylate proteins, compared to other monosaccharides.
- Glucose has a strong tendency to exist in the ring conformation (is the most stable hexose), and therefore tends not to modify proteins. In the ring conformation, all hydroxyl groups are equatorial.
In eukaryotic cells, how are glycolytic enzymes organized?
They are organized in supramolecular complexes found in the cytoplasm. This is an arrangement that increases enzyme efficiency by facilitating movement of substrates and products between enzymes. This process is called substrate channeling and prevents the release of any toxic intermediates.
2 stages of glycolysis
- Glucose is trapped and prepared
2. Oxidizes the 3-carbon compounds to pyruvate while generating two molecules of ATP.
What occurs during stage 1 of glycolysis?
The purpose of the initial steps of stage 1 is to trap the glucose in the cell and form a compound that can be readily cleaved into phosphorylated 3 carbon units- this stage will end when fructose is cleaved into the 3 carbon compounds . Glucose is converted into fructose 1,6-bisphosphate.
Hexokinase
An enzyme that traps glucose in the cell and begins glycolysis. It requires requires Mg2+ or Mn2+ as a cofactor, and catalyzes the transfer of the phosphoryl group from ATP to the hydroxyl group on carbon 6 of glucose.
What happens to glucose when it enters the cell?
Upon entering the cell through a specific transport protein, glucose is phosphorylated by ATP to form glucose 6-phosphate.
Why must glucose be phosphorylated as it enters the cell? (2)
- Glucose-6-phosphate can’t pass through the membrane because of the negative charges on the phosphoryl groups, and it isn’t acted on by glucose transporters
- The addition of the phosphoryl group facilitates the metabolism of glucose into 3 carbon molecules with high phosphoryl transfer potential.
Kinases
Enzymes that catalyze the transfer of a phosphoryl group from ATP to an acceptor
How does hexokinase work?
Hexokinase employs substrate-binding induced fit to help exclude water and minimize undesired hydrolysis of ATP. These structural changes in hexokinase are induced by glucose binding to the enzyme.
Why are the structural changes in hexokinase significant? (2)
- The environment around the glucose becomes more nonpolar, which favors a reaction between the hydrophilic hydroxyl group of glucose and the terminal phosphoryl group of ATP.
- The conformational changes enable the kinase to discriminate against water as a substrate- the cleft in hexokinase closes to prevent water from binding to the active site.
Which molecule does glucose 1,6-biphosphate during glycolysis?
Fructose 1,6-bisphosphate.
Phosphoglucose isomerase
The conversion of glucose 6-phosphate to fructose 6-phosphate is catalyzed by phosphoglucose isomerase. The enzyme opens the 6 carbon ring of glucose 6-phosphate, then catalyzes the isomerization of glucose 6-phosphate, then promotes the formation of the 5 membered ring of fructose 6-phosphate. The reaction is readily reversible.
After the isomerization step of glycolysis, what happens?
A second, irreversible phosphorylation step follows. Fructose 6-phosphate is phosphorylated to fructose 1,6-biphosphate, using ATP molecules. The carbohydrate is trapped in the fructose form.
Phosphofructokinase (PFK)
The second phosphorylation reaction of glycolysis is catalyzed by PFK. PFK is an allosteric enzyme- it binds at another site that isn’t the active site. PFK is also an important regulator of glycolysis.
What is the purpose of the phosphorylation of fructose 6-phosphate to form fructose 1,6-biphosphate?
Phosphorylation of the fructose 6-phosphate to fructose 1,6-biphosphate prevents the reformation of glucose 6-phosphate.
To complete stage 1 of glycolysis, fructose 1,6-biphosphate is cleaved into (2)
- Glyceraldehyde 3-phosphate (GAP)
2. Dihydroxyacetone phosphate (DHAP)
Aldolase
An enzyme that catalyzes the cleavage of fructose 1,6-biphosphate into DHAP and GAP. This reaction is readily reversible.
How are DHAP and GAP processed differently?
GAP is on the direct pathway of glycolysis, while DHAP isn’t. Therefore, GAP can be processed to pyruvate to yield ATP, whereas
DHAP cannot.
Triose phosphate isomerase (TPI)
An enzyme that interconverts GAP and DHAP, allowing the DHAP to be further metabolized. TPI is the only glycolytic enzyme for which genetic deficiency in expression can be lethal. It is characterized by hemolytic anemia and neurodegeneration.
What occurs during a TPI reaction?
TPI catalyzes the transfer if a hydrogen atom from carbon 1 to carbon 2, an intramolecular oxidation-reduction. This is an isomerization of ketose into an aldose, and proceeds through an enediol intermediate.
Steps of a TPI reaction (3)
- Glutamate 165 acts as a general base catalyst and removes a proton from C-1 of the substrate to form the enediol intermediate.
- Glutamate 165, now acting as a general acid catalyst, donates a proton to C-2, while histidine 95 removes a proton from C-1.
- The product is formed, and glutamate 165 and histidine 95 return to their initial states.
Which features of TPI enhance its efficiency? (2)
- TPI is a strong catalyst. It accelerates isomerization much more than other enzymes. The ratio is close to the diffusion-controlled limit
- TPI suppresses an undesired side reaction- the decomposition of the enediol intermediate into methyl glyoxal and orthophosphate.
Diffusion-controlled limit
Catalysis takes place every time that the enzyme and the substrate meet. The diffusion-controlled encounter of substrate and enzyme is thus the rate limiting step in catalysis. TPI is an enzyme of a kinetically perfect enzyme.
Why is the decomposition of the enediol intermediate an undesired reaction?
The reaction occurs much faster than the isomerization reaction, but is physiologically useless. Also, methyl glyoxal is a highly reactive compound that can modify the structure and function of biomolecules, including DNA and proteins. TPI is able to prevent the enediol from leaving the enzyme. The active site is kept closed when enediol is present and remains closed until the isomerization is completed.
What is the initial reaction in the second stage of glycolysis?
Conversion of glyceraldehyde 3-phosphate into 1,3-biphosphoglycerate. The product is generated by the oxidation of GAP in a reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase.
Steps for the reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase (2)
- The highly exergonic oxidation of carbon 1 in GAP to a carboxylic acid by NAD+
- The highly endergonic formation of glyceraldehyde 1, 3-
bisphosphate from the acid and orthophosphate.
How are the two reactions in the formation of glyceraldehyde 1,3-biphosphate linked?
These two reactions are linked by the formation of an energy-rich thioester in the active site of glyceraldehyde 3-phosphate dehydrogenase.
What would happen in the formation of glyceraldehyde 1,3-biphosphate reaction without the thioester intermediate?
The second step (formation of acyl phosphate) would have a large activation barrier and the reaction would be very slow.
Reaction mechanism of glyceraldehyde 3-phosphate
dehydrogenase (4 steps)
- GAP reacts with a cysteine residue to form a
hemithioacetal. - A thioester is formed by the transfer of a hydride to NAD+.
- NADH is exchanged for NAD+. The charge on NAD+
facilitates the attack by the phosphate on the thioester. - Phosphate attacks the thioester, forming the product 1,3-
BPG.
Why must oxidation and acyl-phosphate formation be coupled?
The two processes must be coupled with a thioester intermediate so the favorable aldehyde oxidation can be used to drive the formation of the acyl phosphate. The thioester intermediate is higher in free energy than the free carboxylic acid is, and it preserves much of the free energy released in the oxidation reaction.
1,3- Bisphosphoglycerate
An energy rich molecule with a greater phosphorylation potential than that of ATP. Therefore, 1,3-BPG can be used to power the synthesis of ATP from ADP.
Phosphoglycerate kinase
Catalyzes the transfer of the phosphoryl group from the acyl phosphate of 1,3-bisphosphoglycerate to ADP, ATP and 3-phosphoglycerate are the products.
Substrate-level phosphorylation
The formation of ATP through the transfer of a phosphate group from the acyl phosphate of 1,3-bisphosphoglycerate to ADP. The phosphate donor, 1,3-BPG, is a substrate with high phosphoryl transfer potential.
What are the outcomes of the reactions catalyzed by glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase? (3)
- Glyceraldehyde 3-phosphate, an aldehyde, is oxidized to 3-phosphate, a carboxylic acid
- Following step 1, NAD+ is reduced to NADH
- ATP is formed from Pi and ADP at the expense of carbon-oxidation energy
What happens to the energy released during the oxidation of glyceraldehyde 3-phosphate?
The energy is temporarily trapped as 1,3-bisphosphoglycerate. The energy powers the transfer of a phosphoryl group from 1,3-bisphosphoglycerate to ADP to yield ATP.
Which molecules are converted into glycolytic intermediates?
Fructose, from table sugar or high fructose corn syrup,
and galactose, from milk sugar, can be converted into
glycolytic intermediates.
How is fructose metabolized in the liver?
In the liver (the main site of fructose metabolism), fructose is metabolized by the fructose 1-phosphate pathway
How is fructose metabolized in adipose tissue?
In other tissues (e.g., adipose tissue), fructose is directly
phosphorylated by hexokinase
Excessive fructose consumption can cause (4)
Fructose is a commonly used sweetener. Excess consumption of fructose has been linked to fatty liver, insulin insensitivity, obesity, and type 2 diabetes. In the liver, fructose metabolism bypasses the key regulatory enzyme phosphofructokinase- the excess pyruvate is converted into acetyl CoA and then into fatty acids.
Which molecule is galactose converted into?
Galactose is converted into glucose 6-phosphate by the galactose-glucose interconversion pathway, which begins with the phosphorylation of galactose by galactokinase
Phosphoglucomutase
Glucose 1-phosphate can be converted into glucose 6-
phosphate by phosphoglucomutase.
Lactose intolerance (hypolactasia)
This occurs because most adults lack lactase, the enzyme that degrades lactose. Northern Europeans have a mutation that prevents the decline of lactase activity after weaning. In lactase-deficient individuals, gut bacteria ferment lactose to lactic acid, also generating methane (CH4) and hydrogen gas (H2); the products cause discomfort and disrupt water balance in the intestine.
When does galactose become toxic?
When the transferase is missing
Classic galactosemia
Results if galactose 1-phosphate uridyl transferase activity is deficient. The most common treatment is to remove galactose
(and lactose) from the diet
Classic galactosemia symptoms (4)
Failure to thrive, jaundice, and liver enlargement that can lead to cirrhosis. Cataract formation may also occur
What causes cataracts to form?
Cataracts, a clouding of the lens, form because galactose is converted into galactitol, which is poorly metabolized and accumulates in the lens. Water diffuses into the lens to maintain osmotic balance, causing cataract formation.
In glycolysis, which enzymes are potential control sites? (3)
- Hexokinase,
- Phosphofructokinase
- Pyruvate kinase.
How is phosphofructokinase inhibited and stimulated?
The enzyme is allosterically inhibited by ATP and
allosterically stimulated by AMP. ATP binding lowers the enzyme’s affinity for fructose 6-phosphate, while AMP reverses the inhibitory action of ATP. This means that the activity of the enzyme increases when the ATP/AMP ratio is lowered (i.e., glycolysis is stimulated as the energy charge falls. It’s also inhibited by low pH.
How is phosphofructokinase inhibited by low pH?
Low pH also inhibits phosphofructokinase activity by enhancing the
inhibitory activity of ATP. pH can fall when muscle is functioning
anaerobically, producing excessive amounts of lactic acid, and this
inhibition protects the muscle from damage that would result from
accumulation of too much acid.
Why is AMP, not ADP, the positive regulator of phosphofructokinase?
When ATP needs are great, adenylate kinase can generate ATP from 2 ADP. AMP is released in this reaction, and it then becomes a signal of a low energy state. Also, use of AMP as an allosteric regulator provides sensitive control, because in the cell [ATP] > [ADP] > [AMP]; so small fluctuations in ATP concentration are
magnified into large changes in [AMP] concentration.
Which enzymes are allosterically regulated in glycolysis? (3)
- PFK
- Hexokinase
- Pyruvate kinase
How is hexokinase allosterically regulated?
Hexokinase is allosterically inhibited by its product
glucose 6-phosphate
How is pyruvate kinase allosterically regulated?
Pyruvate kinase is allosterically inhibited by signals ATP and is stimulated by the phosphofructokinase product fructose 1,6-bisphosphate in an example of feedforward stimulation.
What are the main regulators of phosphofructokinase in the liver? (2)
- Citrate
2. Fructose 2,6-bisphosphate
Citrate
One of the main regulators of PFK in the liver- reports on the status of the citric acid cycle. Citrate inhibits phosphofructokinase, whereas fructose 2,6-bisphosphate is a powerful activator.
Glucokinase
The hexokinase isozyme in liver that is primarily responsible for phosphorylating glucose. Glucokinase is active only after a meal, when blood-glucose levels are high, and it is not inhibited by glucose 6-phosphate. The glucokinase isoform has a lower affinity for glucose than the other hexokinases, so it operates only when glucose is abundant.
Glucokinase function
The role of glucokinase is to provide glucose 6-phosphate for the synthesis of glycogen and for the formation of fatty acids
In the liver, how are hexokinase and pyruvate kinase regulated?
Allosterically
How is the liver isozyme of pyruvate kinase regulated?
The liver isozyme of pyruvate kinase is also regulated by covalent modification. Low blood glucose leads to the phosphorylation and inhibition of liver pyruvate kinase. Glucagon triggers this phosphorylation, which prevents the liver from consuming glucose when it is more urgently needed by the brain and muscle.
How does glucose enter and leave animal cells?
Five glucose transporters, known as GLUT1–5, facilitate the movement of glucose across the cell membrane
GLUT 1
Glucose transporter found in all mammalian tissues, responsible for basal glucose intake.
GLUT 2
Found in liver and pancreatic cells. In the pancreas, it plays a role in the regulation of insulin. In the liver, it removes excess glucose from the blood.
GLUT 3
Found in all mammalian tissues, responsible for basal glucose uptake.
GLUT 4
Found in muscle and fat cells. Amount in muscle plasma membrane increases with endurance training.
GLUT 5
Found in the small intestine, primarily a fructose transporter.
Km
The concentration of substrate when an enzyme is active at half of its maximum velocity. The lower the Km, the higher the enzyme’s affinity for the substrate. GLUT 2 has the highest Km, and GLUT 4 has the next highest.
Aerobic glycolysis
Rapidly growing tumors obtain ATP by metabolizing glucose to lactate even in the presence of oxygen. Aerobic glycolysis is a property of rapidly growing cells.
How does aerobic glycolysis facilitate tumor growth? (3)
- The acidic environment that results from the production of lactate has been shown to facilitate tumor invasion.
- Tumors tend to grow in hypoxic environments, since they are not well supplied by blood vessels, so aerobic glycolysis makes them less dependent on oxygen supply.
- In addition, the increased uptake of glucose and formation of glucose 6-phosphate provides substrates for the pentose phosphate pathway, which generates biosynthetic reducing power, NADPH.
How can tumors be visualized?
When patients are infused with a non-metabolizable analog of glucose (FDG), tumors are readily visualized by tomography. This takes advantage of the aerobic glycolysis property of cancer cells.
Hypoxia-inducible transcription factor (HIF-1)
Transcription factor that facilitates aerobic glycosis. The hypoxia that some tumors experience with rapid growth activates this transcription factor. This adaptation allows the tumor to survive until blood vessels can grow. Anaerobic exercise training (forcing muscles to rely on lactic acid fermentation for ATP production) also
stimulates HIF-1, enhancing an athlete’s ability to generate ATP anaerobically and stimulates new blood vessel growth.
Proteins in glucose metabolism encoded by genes regulated by hypoxia-inducible factor (9)
- GLUT 1
- GLUT 3
- Hexokinase
- PFK
- Aldolase
- Glyceraldehyde 3-phosphate dehydrogenase
- Phosphoglycerate kinase
- Enolase
- Pyruvate kinase
Major precursors for gluconeogenesis (3)
Lactate, amino acids, and glycerol
Major sites of gluconeogenesis
The major site of gluconeogenesis is the liver, although some gluconeogenesis can occur in the kidney.
When is gluconeogenesis especially important?
Gluconeogenesis is especially important during fasting or starvation, as glucose is the primary fuel for the brain and the only fuel for red blood cells.
The formation of phosphoenolpyruvate from pyruvate
requires which two enzymes?
Pyruvate carboxylase and phosphoenolpyruvate carboxykinase
Pyruvate carboxylase
Lengthens pyruvate into oxaloacetate, using a molecule of ATP. Also regulates gluconeogenesis. Pyruvate carboxylase uses the vitamin biotin as a cofactor
Reaction mechanism of pyruvate carboxylase (3 stages)
- The biotin carboxylase domain catalyzes the formation
carboxyphosphate. - The carboxylase then transfers the CO2 to the biotin carboxyl
carrier protein (BCCP). - The BCCP carries the activated CO2 to the pyruvate
carboxylase domain, where the CO2 is transferred to pyruvate
Which molecule is a required cofactor for carboxylation of biotin?
Acetyl CoA
The formation of oxaloacetate by pyruvate carboxylase
occurs in
The mitochondria
Oxaloacetate
Oxaloacetate is reduced to malate and transported into the cytoplasm, where it is reoxidized to oxaloacetate with the generation of cytoplasmic NADH. PEP is then synthesized from oxaloacetate by phosphoenolpyruvate carboxykinase.
What is the final step in gluconeogenesis?
The generation of free glucose, which occurs essentially
only in liver, is the final step in gluconeogenesis
How does the generation of free glucose occur?
Glucose 6-phosphate is transported into the lumen of the endoplasmic reticulum. It is an integral membrane on the inner surface of the endoplasmic reticulum, catalyzes the formation of glucose from glucose 6-phosphate. In tissues that do not dephosphorylate glucose, glucose 6-phosphate is converted into glycogen for storage
Why are gluconeogenesis and glycolysis reciprocally regulated?
The rationale for reciprocal regulation is that glycolysis will predominate when glucose is abundant and gluconeogenesis will be highly active when glucose is scarce. If ATP is required, glycolysis predominates. If glucose is required, gluconeogenesis is favored.
Which factors regulate glycolysis and gluconeogenesis?
The interconversion of fructose 1,6-bisphosphate and fructose 6-phosphate is a key regulatory point. In addition, glycolysis and gluconeogenesis are reciprocally regulated at the interconversion of
phosphoenolpyruvate and pyruvate.
How is glucose metabolism in the liver regulated?
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.
Bifunctional enzyme
The kinase that synthesizes fructose 2,6-bisphosphate and the phosphatase that hydrolyzes this molecule are located on the same polypeptide chain. Such an arrangement is called a bifunctional enzyme. Phosphorylation of the bifunctional enzyme activates the
phosphatase activity and inhibits the kinase activity.
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 is called a substrate cycle. Both reactions are not fully active at the same time because of reciprocal allosteric controls
How are substrate cycles biologically important?
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.
Cori cycle
Muscle and liver display interorgan cooperation in a series of reactions called the Cori cycle. Lactate produced by muscle during contraction is released into the blood. Liver removes the lactate and converts it into glucose, which can be released into the blood.
Which parts of the glycolysis and gluconeogenesis pathways are the same?
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
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. 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.
How does TPID impact ATP production?
Because of the enzyme’s (TPI) 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
How does TPID impact protein function?
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.
What are the 2 main features of pyruvate carboxylase deficiency (PCD)
- Hypoglycemia
2. Lactic acidosis
How does PCD cause hypoglycemia?
Hypoglycemia results from the inability to perform gluconeogenesis due to the missing pyruvate carboxylase
How does PCD cause acidosis?
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).
Glucose is converted into fructose 1,6-bisphosphate in 3 steps: phosphorylation, isomerization, and a second phosphorylation reaction
- Phosphorylation
- Isomerization
- Second phosphorylation reaction
