sakai-Glycolysis Flashcards
What is aerobic glycolysis and what is anaerobic glycolysis?
Aerobic glycolysis starts with glucose and ends with pyruvate. It takes place in oxygenated cells with mitochondria that can reoxidize NADH. NADH is formed in glycolysis by glyceraldehyde 3-phosphate dehydrogenase. Cytosolic NADH participates in the malate-aspartate shuttle and the glycerol phosphate shuttle.
Anaerobic glycolysis also starts with glucose but ends with lactate. It takes place in cells without mitochondria (RBC), in cells with insufficient oxygen supply (lens, cancer cells), or in cells during ischemia or anoxia. It is also formed in muscle during intense contraction.
Name the two enzymes that are used for ATP formation by substrate level phosphorylation in glycolysis
Substrate level phosphorylation is performed in cytosol in glycolysis
by phosphoglycerate kinase, a reversible reaction from 1,3 bisphosphoglycerate to 3-phosphoglycerate
[the enzyme is named in the reverse direction of glycolysis]
by pyruvate kinase, an irreversible reaction from PEP to pyruvate.
[misleading name, as pyruvate kinase cannot phosphorylate pyruvate to PEP
inside of cells and this is an irreversible reaction. The formation of PEP from
pyruvate during gluconeogenesis needs several enzymes]
1,3 bisphosphoglycerate is formed in glycolysis. What is the importance of 2,3-bisphosphoglycerate in RBC?
Only RBC contain a high amount (about 5 mmol/L) of 2,3-BPG.
Its concentration is approximately that of hemoglobin. 2,3-BPG is needed for the formation of the T-state of hemoglobin which delivers oxygen to tissues.
The synthesis of 2,3-BPG in RBC is increased at high altitudes in healthy individuals. It is also increased in patients with chronic obstructive pulmonary disease (COPD) and in patients with pyruvate kinase deficiency (due to a backup of glycolytic intermediates).
When you form 2,3-bisphosphoglycerate in RBC, can you perform both steps of substrate level phosphorylation of glycolysis starting with one molecule of glucose? Explain.
The first step of substrate level phosphorylation is catalyzed by phosphoglycerate kinase which needs the energy-rich 1,3-bisphosphoglycerate as substrate.
In the RBC, some of the 1,3 BPG is used in glycolysis for substrate level phosphorylation and some of the molecules are branched out in order to form 2,3-BPG.
2,3-BPG is cleaved to 3-phosphoglycerate which is again a metabolite of glycolysis and leads to PEP which can be used for substrate level phosphorylation.
Overall, some ATP formation from 1,3-BPG is lost, but 2,3-BPG is absolutely necessary for the purpose of RBC to deliver oxygen to tissues.
This shows the importance of pyuvate kinase in RBCs. Hereditary deficiency of pyruvate kinase catalyzing this second step of substrate phosphorylation leads to hemolysis due to lack of ATP.
Name the three irreversible reactions of glycolysis
Glucose and ATP to glucose 6-P and ADP catalyzed by hexokinase (glucokinase is found in liver and in -cells of pancreas)
Fructose 6-P and ATP to fructose 1,6-bisphosphate and ADP catalyzed by phosphofructokinase-1
PEP and ADP to pyruvate and ATP catalyzed by pyruvate kinase
Which glycolytic enzyme needs NAD+ and generates NADH in glycolysis?
Glyceraldehyde 3-P dehydrogenase needs NAD+ and uses inorganic phosphate in order to form 1,3-bisphosphoglycerate.
NADH is generated and can be used in the malate-aspartate shuttle or in the glycerophosphate shuttle when oxygen and mitochondria are available.
NADH is also used to form lactate in anaerobic glycolysis.
Compare hexokinase and glucokinase to each other related to their affinity for glucose and discuss their different purpose. How are they regulated?
Hexokinase has a high affinity for glucose (small Km, much smaller than fasting blood glucose levels) which is important for the metabolism especially in RBC and the brain.
[RBC and brain contain many high affinity glucose transporter GLUT-1]
The purpose of hexokinase is to phosphorylate glucose at normal (or even lower) blood glucose levels when it is needed for the cells. At high levels of glucose 6-P, hexokinase is inhibited by its own product and the glucose is left in the blood for other cells.
Glucokinase has a low affinity for glucose (large Km, about 6-10 mM) and it has a high Vmax and can phosphorylate large amounts of glucose.
The purpose of glucokinase is to phosphorylate as much glucose as possible at high blood glucose levels and to trap glucose 6-P inside the hepatocyte after a meal.
Glucokinase is not product inhibited, it is regulated by the glucokinase regulatory protein (GKRP).
At high fructose 6-P levels in the liver, glucokinase is translocated into the nucleus.
At high cytosolic free glucose levels, glucokinase is transported back into the cytosol and is able to phosphorylate large amounts of glucose.
[note: very unusual regulation]
Name the two cell types that contain glucokinase and discuss the purpose. What happens in hereditary glucokinase deficiency?
Glucokinase is found in hepatocytes where it has the purpose to reduce high blood glucose levels after a carbohydrate-rich meal. GLUT-2 allow uptake of large amounts of glucose from the portal vein into the hepatocyte.
Hereditary deficiency of glucokinase leads to a rare form of diabetes, MODY 2.
Glucokinase is found in -cells of pancreas where it acts as sensor of high blood glucose levels which leads to insulin release. GLUT-2 allow uptake of large amounts of glucose at high blood glucose levels.
Which enzyme catalyzes the committed step of glycolysis?
The committed step of glycolysis is catalyzed by phosphofructokinase-1.
This enzyme is a kinase, it phosphorylates something using ATP, and in this case it phosphorylates an already phosphorylated fructose. 1 indicates that it phosphorylates fructose 6-P to fructose-1,6-bisphosphate.
The pathway of glycolysis is also regulated by hexokinase (glucokinase) but the formed glucose 6-P can still be used in some cells in the HMP or for glycogen synthesis.
PFK-1 forms in the committed step of glycolysis fructose 1,6-bisphosphate which is a metabolite in glycolysis but some molecules bind allosterically to pyruvate kinase in hepatocytes, RBC and proliferating cells and act as allosteric positive heterotropic effector (feed-forward activator).
[note: only fructose 1,6-BP can bind to pyruvate kinase and act as allosteric forward-activator, whereas fructose 2,6-BP cannot. Binding of the allosteric positive heterotropic effector leads to a conformational change of pyruvate kinase and the affinity for PEP now is higher, the K 0.5 is smaller]
Describe the Cori cycle. What is the purpose, what is released into the blood by muscle and what is released by the liver?
The Cori cycle describes the release of lactate from anaerobic glycolysis of active skeletal muscle (and of RBC) into the blood and the uptake of lactate into the liver for gluconeogenesis.
The liver can perform gluconeogenesis and can cleave the generated glucose 6-P to free glucose which can then be released via GLUT-2 into the blood. The generated glucose can be taken up by muscle (or RBC).
[note: the original Cori-cycle described only the interaction between muscle and liver, now often the release of lactate and uptake of glucose by RBC are included when the Cori-cycle is described]
The purpose is to allow formation of lactate in the muscle and RBC and its release into the blood without leading to lactic acidemia. At the same time the valuable lactate is used to generate glucose via gluconeogenesis in the liver.
[note, lactate from the blood is mainly taken up by the liver and the heart. The liver forms pyruvate, which can be used for energy metabolism or in case of fasting, for glucoengenesis. The heart also forms pyruvate but then it is always used for energy metabolism]
Compare the usage of glycolysis in RBC to the usage of glycolysis in the brain!
RBC have no mitochondria and use anaerobic glycolysis for ATP formation.
[note: RBC are always dependent on glucose, also during prolonged fasting. Also, glucose 6-P branches out of glycolysis into the PPP for NADPH formation , and 1,3 BPG branches out for formation of 2,3 BPG. Both “side pathways” join the glycolysis again].
The brain uses glucose for aerobic glycolysis, followed by pyruvate DH complex and TCA cycle.
[note: During prolonged fasting, the brain uses ketone bodies for energy generation but also needs uptake of some glucose for its specific metabolism].
Compare the usage of glycolysis in the liver to muscle!
The healthy liver is always well oxygenated and uses aerobic glycolysis and PDH and TCA cycle. Glycolysis allows the reduction of high blood glucose levels, the liver contains GLUT-2 and glucokinase.
In case that the blood glucose level is still high, the liver continues with the pathways of fatty acid de novo synthesis and cholesterol synthesis.
The muscle uses glycolysis for ATP formation and links glycogen degradation to glycolysis and pyruvate formation. Glycolysis in muscle can be aerobic or anaerobic depending on oxygen supply. Lactate formed at intense muscle contraction is released into the blood.
Describe glycolysis in the lens and in cancer cells.
The cells of the lens and cancer cells are both deprived of oxygen supply.
They use anaerobic glycolysis for ATP formation.
[note: method for detection of cancer growth: a glucose analog can be used to positron emission tomography (PET) to monitor the growth of cancer cells which are dependent on glycolysis for their energy needs]
Which glycolytic enzyme cannot form its normal product when pentavalent arsenic is present? What is the mechanism?
Pentavalent arsenic (arsenate) interferes with glyceraldehyde 3-phosphate dehydrogenase which uses NAD+ and inorganic phosphate.
Arsenate competes with inorganic phosphate, and instead of formation of 1,3-bisphosphoglycerate using inorganic phosphate, arsenate is used and leads now to an instable molecule that is spontaneously hydrolyzed to 3-phosphoglycerate.
With that, the substrate level phosphorylation using the energy-rich 1,3-bisphosphoglycerate cannot be performed in glycolysis.
Which enzyme is inhibited by fluoride ions?
Fluoride ions inhibit enolase which uses 2-phosphoglycerate and forms PEP in glycolysis. This inhibitor can be used in vials for blood collection and prevents glucose usage by RBC while they are in the already drawn blood.
