number 12 Done by Baraa Ayed Corrected by Mamoon Mohammad Alqtamin Doctor Nayef Karadsheh Lactate production 1 P a g e
Advantages of producing lactate Lactate is produced anaerobically to meet the following demands: 1) Cells with low energy demands For example, RBCs which don t have mitochondria cannot undergo oxidative phosphorylation. So, these cells need low amount of energy, and this can be achieved by anaerobic glycolysis and lactate is the product of this pathway. Reduction to lactate is the major fate for pyruvate in tissues that are poorly vascularized (the lens and cornea of the eye, and the kidney medulla). 2) To cope with increased energy demands in vigorously exercising muscles In exercising skeletal muscle, consuming oxygen increases ina way there is no enough amount of oxygen to undergo oxidative phosphorylation to produce enough energy.so, muscles undergo anaerobic glycolysis to provide the required energy. Therefore during intense muscle exercising, lactate accumulates in the muscle. 3) During Hypoxia When the blood supply of oxygen is low, oxygen in tissues is low.so, cells undergo anaerobic glycolysis as a solution to keep cells alive until the blood supply of oxygen is restored. The disadvantage of producing lactate: Lactate acidosis Lactic acid accumulation causes lactate acidosis, whichis the most common cause of metabolic acidosis, and that is due to: 1) Increased production of lactic acid 2) Decreased utilization of lactic acid This leads to: 1) Increased lactic acid concentration above 5mM, whereas the normal concentration (without lactate acidosis) is in the range of(0.4-1.8 mm) 2 P a g e
2) Decreased phthat reaches 7.2 or below, whereas the normal ph is in the range of(7.35-7.45). Common causes of lactate acidosis are: 1) Impaired oxidative phosphorylation"most common" due to: A. Impaired oxygen transport: ex, Myocardial infarction B. Respiratory failure: ex, pulmonary embolism these two points mean that collapse in the circulatory system results in the failure to bring adequate amount of oxygen to the tissues C. Direct inhibition of oxidative phosphorylation:as in increasing the concentration of NADH and cyanide poisoning. D. Uncontrolled hemorrhage: lyses of RBCs 2) Alcohol intoxication:alcohol is rich of NADH, so the NADH/NAD+ ratio is high. This leads to the conversion of pyruvate into lactate by taking two electrons from NADH. Pyruvate +NADH + H+ lactate + NAD+ Rare causes of lactate acidosis: 1- Decrease in gluconeogenesis, which causes accumulation of lactate. 2- Decrease in pyruvate dehydrogenase activity. 3- Decrease in the citric acid cycle activity. 4- Decrease in pyruvate carboxylase activity. This means that there is a deficiency in these enzyme, which is very rare in our bodies. NOTE: the direction of LDH reaction depends on the relative intracellular concentrations of pyruvate and lactate and the ratio of NADH/NAD+. In the liver and heart this ratio is lower than exercising muscle. Consequently the liver and heart oxidize lactate (obtained from the blood) to pyruvate. 3 P a g e
The inorganic inhibitors of glycolysis 1) Fluoride inhibits enolase enzyme: fluoridated water inhibits bacterial enolase,which prevents dental caries so it is used in toothpastes. 2) Arsenic poisoning:there are Two forms of arsenic compound Arsenate (pentavalent) Arsenite (trivalent) It's not as strong as arsenite. It prevents ATP and NADH production (at the level of glyceraldehyde 3-phosphate), therefore it competes with Pi as a substrate for G3P dehydrogenase. So, it will form 1-arseno-3- phosphoglycerate rather than 1, 3- Bisphosphoglycerate d More harmful than arsenate because it forms stable complex in which it binds to SH group in lipoic acid. As you know lipoic acid is a cofactor in oxidative decarboxylase enzymes (α ketoglutarate dehydrogenase complex, pyruvate dehydrogenase complex). This leads to inactivate these enzymes and cause neurological diseases and death. Regulation of glycolysis In glycolysis we have 10 steps, 3 of them are irreversible steps and this is where regulation takes place; The first step is the phosphorylation of glucose The second step is adding a phosphate group to fructose 6-P to form fructose 1, 6-bisphosphate (this is the most important step to be regulated) 4 P a g e
The last step is the conversion of phosphoenolpyruvate to pyruvate. Regulation of the first step (Phosphorylation of Glucose) by allosteric hormonal regulation ALLOSTERIC: Phosphorylation of glucose is done either by hexokinase that is found in all tissues and involved in the use of glucose for energy production, or byglucokinase that is found in the liver and isinvolved in the storage of glucose. Remember: glucokinase has high Km, low affinity for glucose, it needs high concentration of glucose to be activated, and it has high Vmax. WhereasHexokinase has low km, high affinity for glucose, it needs very low concentration of glucose to be activated, and it has low Vmax. HORMONAL: these enzymes are affected by hormones such as insulin and glucagon. NOW, we will discuss the mechanism of regulation for hexokinase and glucokinase: Hexokinase is inhibited by its product, glucose 6- phosphate. When glucose 6-phosphate accumulates, hexokinase becomes less efficient. Glucokinase is inhibited by fructose 6-phosphate rather than glucose 6-phosphate. When fructose 6-phosphate accumulates, it promotes glucokinase to be translocated to the nucleus, where it binds to glucokinase regulatory protein (GKRP). So, it becomes an inactive complex. When the concentration of glucose is high, it will induce secreting glucokinase from its complex in the nucleus,glucokinasegoes back to the cytosol and is activated to phosphorylate glucose to glucose 6 phosphate to be used to form pyruvate, oranabolize to form glycogen. 5 P a g e
Insulin is called"insulin anabolic hormones" When (insulin/glucagon) ratio is high this induces and increases the amount ofglucokinase. This step is not completely irreversible, but highly irreversible. NOTE: when the blood sugar is low, glucagon is secreted and lead to the degradation of glycogen. Regulation of Phosphofructokinase, the second irreversible step Fructose 6-phosphate is phosphorylated to fructose 1, 6- bisphosphate by phosphofructokinase. This allosteric enzyme is activated by 1. Fructose 2, 6bisphosphate(very important activator) 2. AMP (high AMP means that the cell needs energy and glycolysis must be activated 3. When (insulin/glucagon) ratio is high this induces the activity of the enzyme and increases its number. It is inhibited by ATP (when ATP concentration is high, there is No need for glycolysis, citrate and protons). There are two coupled enzymes in our body: phosphofructokinase-1 (PFK1) and phosphofructokinase- 2(PFK2). PFK1 converts fructose 6 phosphate to fructose 1, 6phosphate and PFK2 converts fructose 6 phosphate to fructose 2, 6 phosphate (the most potent activator for PFK1). PFK2 has two domains (bi-functional protein) that has both: 1. Kinase activity that produce fructose 2, 6 phosphate 2. Phosphatase activity that dephosphorylates fructose 2, 6 phosphate back to fructose 6 phosphate. Note: (in the liver, the kinase domain is active if dephosphorylated, and inactive if phosphorylated). So, when a phosphate group is added to PFK-2 by protein kinase A, the 6 P a g e
kinase is inactivated and the phosphatase is activated and vice versa. We said that fructose 2,6 phosphate is an activator for PFK1; when it accumulates it activates PFK1. Therefore glycolysis is activated and gluconeogenesis is inhibited. When hyperglycemia happens, insulin levels are high and insulin binds to its receptors. The signal from this binding is transmitted inside of the cell to activate a special protein which is protein phosphatase, to dephosphorylate the PFK-2. Dephosphorylation of PFK-2, thus, activates the kinase and inactivates phosphatase activity (this enzyme has two activities). So, glycolysis is activated due to the increase of the production of Fructose-2, 6-bisphosphate, because this compound will activate the PFK-1 which produces fructose- 1,6- bisphosphate When hypoglycemia happens (like in fasting, glucagon level is highand it binds to its receptors leading to the activation of adenylylcyclase and increasing camp. Protein kinase A is activated which favors phosphorylation of PFK-2. Phosphorylation of PFK-2, thus, deactivates the kinase and activates phosphatase activity. Note that high glucagon levels indicate the scarcity of glucose, so breaking down glucose must be reduced and gluconeogenesis must be activated. (glucose should be preserved). 7 P a g e
a)regulation of PFK by AMP, ATP and fructose 2, 6 phosphate: The allosteric activators of PFK1 are fructose 2, 6 phosphateand AMP. When AMP level is high, this means that the energy charge of the cell is low. So, the cell needs energy and AMP activates glycolysis which is an energy yielding process. Theseeffectors shift the curve to the left and the curve becomes hyperbolic. The enzyme is much more active. When we increase ATP, the activity initially increases but at higher ATP concentration, ATP binds to allosteric regulatory sites in the enzyme and inhibits it. As you see in this curve, the 8 P a g e
activity of the enzyme suddenly decreases at higher ATP concentration.amp and fructose 2, 6-bisphosphate relieve the inhibition of PFK. Note: AMP and fructose 2, 6 bisphosphate are deinhibitors of ATPand allosteric activators of the enzyme....... During rest, ATP concentration is 5mM. During exercise, ATP level is decreased in a small amount to reach almost 4 Mm. But these two concentrations of ATP are inhibitors. The question is what induces glycolysis during exercising? Note that AMP also increases so it relieves the inhibition of glycolysis by ATP....... b) Regulation of PFK1 by citrate: When hypoglycaemia happens, this reaction happens: oxaloacetate + acetyl-coa Citrate Citrate increases when there is low glucose concentration (hypoglycaemia), and it inhibits PFK1 to slow down glycolysis because we get energy from acetyl-coa. Increasing citrate levels also induces mobilizing the fat to provide a supply of glucose. This glucose that results from fat mobilizing (gluconeogenesis). Glucose is preserved forcritical tissues that only benefit from glucose, like the brain and RBCs. Regulation of Pyruvate Kinase, the third irreversible step. 9 P a g e
This enzyme is activated by fructose 1, 6- bisphosphate, by feed-forward activation. Remember that fructose 1, 6- bisphosphate is an intermediate result from activation of PFK1. Pyruvate kinase is inhibited by ATP and the amino acid alanine becausealanin is produced during glyconeogenesis (mobilization of fat, protein...) which is an indication of low glucose levels. So, we should inhibit glycolysis in order to preserve glucose. When we have hypoglycemia; alanine is an amino acid that is converted to pyruvate then to glucose. Pyruvate kinase (in the liver only) is also regulated by phosphorylation and dephosphorylation. When it is phosphorylated, it is inactive and when it is dephosphorylated, it is active. It is converted from the active form to the inactive form by phosphorylation,catalysed by protein kinase A, which is activated by camp, which is increased by glucagon hormone. Glucagon hormone is an indication of low glucose levels so it deactivates pyruvate kinase to inhibit glycolysis. We finished glycolysis regulation...... Glycolytic enzymes deficiencies When there is deficiency of glycolytic enzymes, ATP production decreases. ATP is necessary for RBCs to keep ion pumps working to maintain the flexible shape of the cell. So, reduction of energy supply will affect its flexible structure and the morphology of the cell (In order to prevent RBCs from not being able to enter the narrow capillaries to pass and to prevent haemolysis).also, there will be shortage in the half-life of the cell. All glycolytic enzymes deficiencies are observed but they are extremely rare. Pyruvate kinase deficiency is the most common among the glycolytic enzymes deficiencies (95%). Diagnosis of pyruvate kinase deficiency is hard because the 10 P a g e
deficiency might be in the amount of it or in the properties of it.the enzyme might show an abnormal response/sensitivity to the activator fructose 1, 6-bisphosphate. Also,the presence of several mutations in the enzyme gene may affect the enzyme.any mutation will change something regarding the enzyme; it may change its kinetics (Km or Vmax)/its amount/ its regulation/its stability. NOTES: In Jordan we observed two cases of pyruvate kinase (PK) deficiency. Diagnosis of pyruvate kinase is hard in the lab because the enzyme is under optimal conditions (substrates, temperature), even though in real life these conditions aren t always found, the enzyme works very well. 11 P a g e