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1 handout biokimia lanjut. p.ukun

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1 handout biokimia lanjut. p.ukun

  1. 1. INTEGRATION OF METABOLISM Metabolism consists of catabolism and anabolism Catabolism: degradative pathways – Usually energy-yielding! Anabolism: biosynthetic pathways – energy-requiring!
  2. 2. Catabolism and Anabolism Catabolic pathways converge to a few end products Anabolic pathways diverge to synthesize many biomolecules Some pathways serve both in catabolism and anabolism Such pathways are amphibolic
  3. 3. Organization in Pathways Pathways consist of sequential steps The enzymes may be separate Or may form a multienzyme complex Or may be a membrane-bound system New research indicates that multienzyme complexes are more common than once thought
  4. 4. Mutienzyme complex Separate enzymes Membrane Bound System
  5. 5. Organization of Pathways Linear (product of rxns are substrates for subsequent rxns) Closed Loop (intermediates recycled) Spiral (same set of enzymes used repeatedly)
  6. 6. Themes in Metabolic Regulation • Allosteric regulation • Covalent modification • Control of enzyme levels • Compartmentalization • Metabolic specialization of organs
  7. 7. Allosteric Regulation  End products are often inhibitors  Allosteric modulators bind to site other than the active site  Allosteric enzymes usually have 4o structure  Vo vs [S] plots give sigmoidal curve for at least one substrate  Can remove allosteric site without effecting enzymatic action
  8. 8. Regulation of Enzyme Activity (biochemical regulation)  1st committed step of a biosynthetic pathway or enzymes at pathway branch points often regulated by feedback inhibition.  Efficient use of biosynthetic precursors and energy B A C 1 3” 3’ 2 E F G 4’ 5’ H I J 4” 5” X X
  9. 9. Vo vs [S] plots give sigmoidal curve for at least one substrate Binding of allosteric inhibitor or activator does not effect Vmax, but does alter Km Allosteric enzyme do not follow M-M kinetics
  10. 10. Pengaruh aktivator ADP terhadap aktivitas enzim Fosfofruktokinase (FPK1)
  11. 11. Allosteric T to R transition Concerted model Sequential model ET-I ET ER ER-S I I S S
  12. 12. Allosteric modulators bind to site other than the active site and allosteric enzymes have 4o structure Fructose-6-P + ATP -----> Fructose-1,6-bisphosphate + ADP ADP Allosteric Activator (ADP) binds distal to active site
  13. 13. Regulation of Hexose Transporters  Intra-cellular [glucose] are much lower than blood [glucose].  Glucose imported into cells through a passive glucose transporter.  Elevated blood glucose and insulin levels leads to increased number of glucose transporters in muscle and adipose cell plasma membranes.
  14. 14. Covalent Modification • Covalent modification of last step in signal transduction pathway • Allows pathway to be rapidly up or down regulated by small amounts of triggering signal (HORMONES) • Last longer than do allosteric regulation (seconds to minutes) • Functions at whole body level
  15. 15. Covalent modification •Regulation by covalent modification is slower than allosteric regulation •Reversible •Require one enzyme for activation and one enzyme for inactivation •Covalent modification freezes enzyme T or R-conformation
  16. 16. Phosphorylation/dephosphorylation •Most common covalent modification •Involve protein kinases/phosphatase •PDK inactivated by phosphorylation •Amino acids with –OH groups are targets for phosphorylation •Phosphates are bulky (-) charged groups which effect conformation
  17. 17. Enzyme Levels • Amount of enzyme determines rates of activity • Regulation occurs at the level of gene expression • Transcription, translation • mRNA turnover, protein turnover • Can also occur in response to hormones • Longer term type of regulation
  18. 18. Regulation of Gene Expression AAAAAA5’CAP mRNA RNA Processing RNA Degradation Protein DegradationPost-translational modification Active enzyme
  19. 19. Compartmentalization One way to allow reciprocal regulation of catabolic and anabolic processes
  20. 20. Metabolic Specialization of Organs
  21. 21. Specialization of Organs • Regulation in higher eukaryotes • Organs have different metabolic roles i.e. Liver = gluconeogenesis, Muscle = glycolysis • Metabolic specialization is the result of differential gene expression
  22. 22. Brain • Glucose is the primary fuel for the brain • Brain lacks fuel stores, requires constant supply of glucose • Consumes 60% of whole body glucose in resting state. Required too maintain Na and K membrane potential in of nerve cells • Fats can’t serve as fuel because blood brain barrier prevents albumin access. • Under starvation can ketone bodies used.
  23. 23. Muscle • Glucose, fatty acids and ketone bodies are fuels for muscles • Muscles have large stores of glycogen (3/4 of body glycogen in muscle) • Muscles do not export glucose (no glucose-6- phosphatase) • In active muscle glycolysis exceeds citric acid cycle, therefore lactic acid formation occurs • Cori Cycle required
  24. 24. Siklus Cori (Cori Cycle)
  25. 25. Muscle • Muscles can’t do urea cycle. So excrete large amounts of alanine to get rid of ammonia (Glucose Alanine Cycle) • Resting muscle uses fatty acids to meet 85% of energy needs
  26. 26. Heart Muscle • Heart exclusively aerobic and has no glycogen stores. • Fatty acids are the hearts primary fuel source. Can also use ketone bodies. Doesn’t like glucose
  27. 27. Liver • Major function is to provide fuel for the brain, muscle and other tissues • Metabolic hub of the body • Most compounds absorb from diet must first pass through the liver, which regulates blood levels of metabolites
  28. 28. Liver: carbohydrate metabolism • Liver removes 2/3 of glucose from the blood • Glucose is converted to glucose-6-phosphate (glucokinase) • Liver does not use glucose as a fuel. Only as a source of carbon skeletons for biosynthetic processes. • Glucose-6-phosphate goes to glycogen (liver stores ¼ body glycogen)
  29. 29. Liver: lipid metabolism • Excess glucose-6-phosphate goes to glycolysis to form acetyl-CoA • Acetyl-CoA goes to form lipids (fatty acids cholesterol) • Glucose-6-phosphate also goes to PPP to generate NADH for lipid biosynthesis • When fuels are abundant triacylglycerol and cholesterol are secreted to the blood stream in LDLs. LDLs transfer fats and cholesterol to adipose tissue. • Liver can not use ketone bodies for fuel.
  30. 30. Liver: protein/amino acid metabolism • Liver absorbs the majority of dietary amino acids. • These amino acids are primarily used for protein synthesis • When extra amino acids are present the liver or obtained from the glucose alanine cycle amino acids are catabolized • Carbon skeletons from amino acids directed towards gluconeogenesis for livers fuel source
  31. 31. Adipose Tissue  Enormous stores of Triacyglycerol  Fatty acids imported into adipocytes from chylomicrons and VLDLs as free fatty acids  Once in the cell they are esterified to glycerol backbone.  Glucagon/epinephrine stimulate reverse process
  32. 32. Well-Fed State • Glucose and amino acids enter blood stream, triacylglycerol packed into chylomicrons • Insulin is secreted, stimulates storage of fuels • Stimulates glycogen synthesis in liver and muscles • Stimulates glycolysis in liver which generates acetyl-CoA for fatty acid synthesis
  33. 33. Refed State • Liver initially does not absorb glucose, lets glucose go to peripheral tissues, and stays in gluconeogenesis mode • Newly synthesized glucose goes to replenish glycogen stores • As blood glucose levels rise, liver completes replenishment of glycogen stores. • Excess glucose goes to fat production.
  34. 34. Starvation  Fuels change from glucose to fatty acids to ketone bodies
  35. 35. GLIKOLISIS  Glikolisis terdiri dari 2 fase: Fase preparasi (preparatory phase), yaitu fosforilasi glukosa dan konversinya menjadi gliseraldehid 3-fosfat. Fase pembayaran (payoff phase), yaitu konversi oksidatif gliseraldehid 3-P menjadi piruvat disertai pembentukan ATP dan NADH.
  36. 36. Preparatory phase
  37. 37. Payoff phase
  38. 38. • Seven steps of glycolysis are retained • Three steps are replaced • The new reactions provide for a spontaneous pathway (G negative in the direction of sugar synthesis), and they provide new mechanisms of regulation
  39. 39. Control Points in Glycolysis
  40. 40. Regulation of Hexokinase  Glucose-6-phosphate is an allosteric inhibitor of hexokinase.  Levels of glucose-6-phosphate increase when down stream steps are inhibited.  This coordinates the regulation of hexokinase with other regulatory enzymes in glycolysis.  Hexokinase is not necessary the first regulatory step inhibited.
  41. 41. Regulation of PhosphoFructokinase (PFK-1)  PKF-1 has quaternary structure  Inhibited by ATP and Citrate  Activated by AMP and Fructose-2,6- bisphosphate  Regulation related to energy status of cell.
  42. 42. Effect of ATP on PFK-1 Activity
  43. 43. Effect of ADP and AMP on PFK-1 Activity
  44. 44. Regulation of fructose 1,6-bisphosphatase-1 (FBPase-1) and phosphofructokinase-1 (PFK-1)
  45. 45. Regulation of PFK by Fructose-2,6-bisphosphate • Fructose-2,6-bisphosphate is an allosteric activator of PFK in eukaryotes, but not prokaryotes •Formed from fructose-6-phosphate by PFK-2 •Degraded to fructose-6-phosphate by fructrose 2,6- bisphosphatase. •In mammals the 2 activities are on the same enzyme •PFK-2 inhibited by Pi and stimulated by citrate
  46. 46. Regulation of Pyruvate Kinase  Allosteric enzyme  Activated by Fructose-1,6-bisphosphate (example of feed-forward regulation)  Inhibited by ATP  When high fructose 1,6-bisphosphate present plot of [S] vs Vo goes from sigmoidal to hyperbolic.  Increasing ATP concentration increases Km for PEP.  In liver, PK also regulated by glucagon. Protein kinase A phosphorylates PK and decreases PK acitivty.
  47. 47. Pyruvate Kinase Regulation
  48. 48. Deregulation of Glycolysis in Cancer Cells  Glucose uptake and glycolysis is ten times faster in solid tumors than in non-cancerous tissues.  Tumor cells initally lack connection to blood supply so limited oxygen supply  Tumor cells have fewer mitochondrial, depend more on glycolysis for ATP  Increase levels of glycolytic enzymes in tumors (oncogene Ras and tumor suppressor gene p53 involved)
  49. 49. Gluconeogenesis • Synthesis of "new glucose" from common metabolites • Humans consume 160 g of glucose per day • 75% of that is in the brain • Body fluids contain only 20 g of glucose • Glycogen stores yield 180-200 g of glucose • The body must still be able to make its own glucose
  50. 50. Gluconeogenesis • Occurs mainly in liver and kidneys • Not the mere reversal of glycolysis for 2 reasons: – Energetics must change to make gluconeogenesis favorable (delta G of glycolysis = -74 kJ/mol – Reciprocal regulation must turn one on and the other off - this requires something new!
  51. 51. • Seven steps of glycolysis are retained • Three steps are replaced • The new reactions provide for a spontaneous pathway (G negative in the direction of sugar synthesis), and they provide new mechanisms of regulation
  52. 52. Regulation of Gluconeogenesis • Reciprocal control with glycolysis • When glycolysis is turned on, gluconeogenesis should be turned off • When energy status of cell is high, glycolysis should be off and pyruvate, etc., should be used for synthesis and storage of glucose • When energy status is low, glucose should be rapidly degraded to provide energy • The regulated steps of glycolysis are the very steps that are regulated in the reverse direction!
  53. 53. Transaminasi The first of the bypass reactions in gluconeogenesis is the conversion of pyruvate to Phosphoenolpyruvate (PEP) Conversion of Pyruvate to Phosphoenolpyruvate Requires Two Exergonic Reactions
  54. 54. Alternative paths from pyruvate to phosphoenolpyruvat e
  55. 55. Pyruvate Carboxylase • The reaction requires ATP and bicarbonate as substrates • Biotin cofactor • Acetyl-CoA is an allosteric activator • Regulation: when ATP or acetyl-CoA are high, pyruvate enters gluconeogenesis
  56. 56. PEP Carboxykinase • Lots of energy needed to drive this reaction! • Energy is provided in 2 ways: – Decarboxylation is a favorable reaction – GTP is hydrolyzed • GTP used here is equivalent to an ATP
  57. 57. PEP Carboxykinase  Not an allosteric enzyme  Rxn reversible in vitro but irreversible in vivo  Activity is mainly regulated by control of enzyme levels by modulation of gene expression  Glucagon induces increased PEP carboxykinase gene expression
  58. 58. Fructose-1,6-bisphosphatase • Thermodynamically favorable - G in liver is - 8.6 kJ/mol • Allosteric regulation: – citrate stimulates – fructose-2,6--bisphosphate inhibits – AMP inhibits
  59. 59. Glucose-6-Phosphatase • Presence of G-6-Pase in ER of liver and kidney cells makes gluconeogenesis possible • Muscle and brain do not do gluconeogenesis • G-6-P is hydrolyzed as it passes into the ER • ER vesicles filled with glucose diffuse to the plasma membrane, fuse with it and open, releasing glucose into the bloodstream.
  60. 60. Regulation of Gluconeogenesis • Reciprocal control with glycolysis • When glycolysis is turned on, gluconeogenesis should be turned off • When energy status of cell is high, glycolysis should be off and pyruvate, etc., should be used for synthesis and storage of glucose • When energy status is low, glucose should be rapidly degraded to provide energy • The regulated steps of glycolysis are the very steps that are regulated in the reverse direction!
  61. 61. •Metabolites other than pyruvate can enter gluconeogenesis •Lactate (Cori Cycle) transported to liver for gluconeogenesis •Glycerol from Triacylglycerol catabolism •Pyruvate and OAA from amino acids (transamination rxns) •Malate from glycoxylate cycle -> OAA -> gluconeogenesis
  62. 62. The Metabolism of Glycogen in Animals Glycogen granules in a hepatocyte
  63. 63. Hormonal Regulation of Glycogen Metabolism Insulin  Secreted by pancreas under high blood [glucose]  Stimulates Glycogen synthesis in liver  Increases glucose transport into muscles and adipose tissues Glucagon  Secreted by pancreas in response to low blood [glucose]  Stimulates glycogen breakdown  Acts primarily in liver Ephinephrine  Secrete by adrenal gland (“fight or flight” response)  Stimulates glycogen breakdown.  Increases rates of glycolysis in muscles and release of glucose from the liver
  64. 64. Metabolism of Tissue Glycogen • But tissue glycogen is an important energy reservoir - its breakdown is carefully controlled • Glycogen consists of "granules" of high MW • Glycogen phosphorylase cleaves glucose from the nonreducing ends of glycogen molecules • This is a phosphorolysis, not a hydrolysis • Metabolic advantage: product is a sugar-P - a "sort-of" glycolysis substrate
  65. 65. •Glycogen phosphorylase cleaves glycogen at non-reducing end to generate glucose-1-phosphate •Debranching of limit dextrin occurs in two steps. •1st, 3 X 1,4 linked glucose residues are transferred to non-reducing end of glycogen •2nd, amylo-1,6-glucosidase cleaves 1,6 linked glucose residue. •Glucose-1-phosphate is converted to glucose-6-phosphate by phosphoglucomutase
  66. 66. The catabolic pathways:  from glycogen to glucose 6-phosphate (glycogenolysis) and from glucose 6-phosphate to pyruvate (glycolysis) The anabolic pathways:  from pyruvate to glucose (gluconeogenesis) and  from glucose to glycogen (glycogenesis)
  67. 67. Glycogen Breakdown Is Catalyzed by Glycogen Phosphorylase
  68. 68. Glycogen Synthase • Forms -(1 4) glycosidic bonds in glycogen • Glycogen synthesis depends on sugar nucleotides UDP-Glucose • Glycogenin (a protein!) protein scaffold on which glycogen molecule is built. • Glycogen Synthase requires 4 to 8 glucose primer on Glycogenin (glycogenein catalyzes primer formation) • First glucose is linked to a tyrosine -OH • Glycogen synthase transfers glucosyl units from UDP-glucose to C- 4 hydroxyl at a nonreducing end of a glycogen strand.
  69. 69. Branch synthesis in glycogen: The glycogen-branching enzyme (also called amylo (1→4) to (1→6) transglycosylase or glycosyl- (4→6)-transferase)
  70. 70. Coordinated Regulation of Glycogen Synthesis and Breakdown  Glycogen Phosphorylase Is Regulated Allosterically and Hormonally  Glycogen Synthase Is Also Regulated by Phosphorylation and Dephosphorylation
  71. 71. Control of Glycogen Metabolism • A highly regulated process, involving reciprocal control of glycogen phosphorylase (GP) and glycogen synthase (GS) • GP allosterically activated by AMP and inhibited by ATP, glucose-6-P and caffeine • GS is stimulated by glucose-6-P • Both enzymes are regulated by covalent modification - phosphorylation
  72. 72. Glycogen phosphorylase of liver as a glucose sensor
  73. 73. Hormonal Regulation of Glycogen Metabolism
  74. 74. Effect of glucagon and epinephrine on glycogen phosphorylase glycogen synthase activities
  75. 75. Effect of insulin on glycogen phosphorylase and glycogen synthase activities
  76. 76. LIPOPROTEIN
  77. 77. Processing of dietary lipids in vertebrates
  78. 78. Lipoproteins  Transport water insoluble TAG, cholesterol and cholesterol-esters throughout circulatory system  Hydrophobic core containing TAG and cholesterol-esters  Hydrophillic surface made of proteins (apoproteins) and phospholipids)
  79. 79. Common membrane phospholipids P O OO O H CH2 H CH2C O O C C OO R1 R2 P O OO O CH2 CH2 H CH2C O O C C OO R1 R2 CH2 NH3 P O OO O CH2 CH2 H CH2C O O C C OO R1 R2 CH2 NH3 COO P O OO O CH2 CH2 H CH2C O O C C OO R1 R2 CH2 NH3C CH3 CH3 Phosphatidate Phosphatidylethanolamine Phosphatidylserine Phosphatidylcholine
  80. 80.  Fatty acids and MAG enter mucosal cells where they are used to re-synthesize TAG  TAG is then packaged into lipoprotein transport particles called chylomicrons (lipoprotein).  Chylomicrons are mainly composed of TAG and apoprotein B-48. Also contain fat solubel vitamins  Chylomicrons enter the lymph system and then the blood stream.  Chylomicrons bind to membrane bound lipoprotein lipases at the surface of adipose and muscle cells.
  81. 81. Lipoproteins  Several different classes of lipoproteins.  Chylomicrons deliver dietary fats to tissues  VLDL, IDL and LDL transport endogenously synthesized TAG and cholesterol to tissues  HDLs remove cholesterol from serum and tissues and transports it back to the liver.  VLDL, IDL, LDL, and HDL named based on their density. Low density lipoproteins have high lipid to protein ratios. High density lipoproteins have low lipid to protein ratios.
  82. 82. Lipoproteins  Lipases in capillaries of adipose and muscle tissues degrade TAG in VLDLs. VLDLs become IDLs.  IDLs can then give up more lipid and become LDLs.  LDLs are rich in cholesterol and cholesterol-esters.
  83. 83. Apolipoproteins  VLDLs, IDLs, and LDLs all contain a large monomeric protein called ApoB-100.  ApoB-100 forms amphipathic crust on lipoprotein surface.  Chylomicrons contain analogous lipoprotein ApoB- 48.  VLDLs and IDLs also possess a number of small weakly associated proteins that disassociate during lipoprotein degradation.  Small apolipoproteins function to modulate the activity of enzymes involved in lipid mobilization and interactions with cell surface receptors.
  84. 84. LDL Receptor  Binds to ApoB-100.  Found on cell surface of many cell types  Mediates delivery of cholesterol by inducing endocytosis and fusion with lysosomes.  Lysosomal lipases and proteases degrade the LDL. Cholesterol then incorporates into cell membranes or is stored as cholesterol-esters.
  85. 85. High LDL levels can lead to cardiovascular disease.  LDL can be oxidized to form oxLDL  oxLDL is taken up by immune cells called macrophages.  Macrophages become engorged to form foam cells.  Foam cells become trapped in the walls of blood vessels and contribute to the formation of atherosclerotic plaques.  Causes narrowing of the arteries which can lead to heart attacks.
  86. 86. Plaque Build up in Artery
  87. 87. Absence of LDL Receptor Leads to Hypercholesteremia and Atherosclerosis  Persons lacking the LDL receptor suffer from familial hypercholesterolemia  Result of a mutation in a single autosomal gene  Total plasma cholesterol and LDL levels are elevated.  Homozygous individuals have cholesterol levels of 680 mg/dL. Heterozygous individuals = 300 mg/dL. Healthy person = <200 mg/dL.  Most homozygous individuals die of cardiovascular disease in childhood.
  88. 88. LDLs/HDLs and Cardiovascular Disease  LDL/HDL ratios are used as a diagnostic tool for signs of cardiovascular disease  LDL = “Bad Cholesterol”  HDL = “Good Cholesterol”  A good LDL/HDL ratio is 3.5  Protective role of HDL not clear.  An esterase that breaks down oxidized lipids is associated with HDL. It is possible (but not proven) that this enzyme helps destroy oxLDL

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