BIOCHEM_LC6_GLUCONEOGENESIS AND PENTOSE PATHWAY.pdf

all the amino acids membrane lipids COURSE OUTLINE nucleotides in DNA and RNA I. REVIEW OF GLY...

all the amino acids membrane lipids COURSE OUTLINE nucleotides in DNA and RNA I. REVIEW OF GLYCOLYSIS and FATES OF cofactors needed for the PYRUVATES metabolism A. Central Importance of Glucose B. Four Major Pathway of Glucose Utilization B. Four Major Pathways of Glucose C. Glycolysis Utilization D. Glycolysis Regulation D.1. Hexokinase Affinity Storage D.2. Glucose Transporters - can be stored in the polymeric D.3. Fructose-2,6 bisphosphate form (starch, glycogen) D.4. Phosphofructokinase 1 D.5. Regulation by Reactants and - used for later energy needs Products Energy production E. Fates of Pyruvate - generates energy via oxidation of II. GLUCONEOGENESIS glucose A. Precursors for Gluconeogenesis B. Glycolysis vs. Gluconeogenesis - short-term energy needs C. Gluconeogenesis Pathway Production of NADPH and pentoses D. Gluconeogenesis Regulator and Key - generates NADPH for use in Process E. Gluconeogenesis from Amino Acids relieving oxidative stress and F. Gluconeogenesis from Glycerol synthesizing fatty acids G. Gluconeogenesis from Propionate - generates pentose phosphates H. Gluconeogenesis from Lactate (Cori for use in DNA/RNA biosynthesis Cycle) III. ANAEROBIC RESPIRATION Structural carbohydrate production A. Anaerobic Glycolysis: Fermentation - used for generation of alternate B. Lactic Acid Fermentation carbohydrates used in cell walls C. Cori Cycle IV. PENTOSE PHOSPHATE PATHWAY of bacteria, fungi, and plants A. Four Major Pathway of Glucose Utilization B. Two Phases of the PPP C. Pentose Phosphate Pathway C.1. Oxidative Phase Generates NADPH and a Pentose D. NADPH and Oxidative Stress E. Nonoxidative Phase Regeneration G-6-P from R-5-P F. PPP Clinical Correlates: G-6PD Deficiency G. Summary V. REFERENCES GLUCONEOGENESIS AND PENTOSE PATHWAY I. GLYCOLYSIS REVIEW A. CENTRAL IMPORTANCE OF Figure 1. Major pathways of glucose utilization GLUCOSE Glucose is an excellent fuel. C. GLYCOLYSIS - yields good amount of energy The glycolytic pathway is employed by all upon oxidation tissues for the oxidation of glucose to -2840 kJ/mol glucose provide energy (in the form of ATP) and - can be efficiently stored in the intermediates for other metabolic polymeric form pathways. - Many organisms and tissues can Pyruvate is the end product of glycolysis. meet their energy needs on What are the reactants and products of glucose only. glycolysis? - Glucose is converted to Glucose is a versatile biochemical pyruvate. precursor. What is the function of glycolysis? - In - Many organisms can use glucose order to derive energy to generate: BIOCHEMISTRY LC6: GLUCONEOGENESIS AND PENTOSE PATHWAY DR. ESPIRITU, A. DATE: 09/19/2024 The first step is an irreversible process Rate limiting step is a VERY SLOW process Last key step is the formation of pyruvate from phosphophenol pyruvate which is also an irreversible process Substrate-phosphorylation the phosphate will come from substrate and will go toward ATP Oxidative-phosphorylation – happens in the electron transport chain D. GLYCOLYSIS REGULATION Figure 2. Phases of glycolysis Used: - 1 glucose; 2 ATP; 2 NAD+ - NAD+ is can be used in the oxygen in the electron transport chain. To produce more ATP later on The glycolysis has a preparatory phase: you need to use ATP first Made: If there are a lot of glucose it will activate - 2 pyruvate glycolysis various different fates D.1. Hexokinase Affinity - 4 ATP Used for energy-requiring processes within the cell ○ 2 NADH Must be reoxidized to NAD+ in order for glycolysis to continue Glycolysis is heavily regulated Hexokinase has higher glucose affinity ○ Ensure proper use of nutrients Km (Michaelis constant) is associated with ○ Ensure production of ATP or, affinity when needed ○ If Km is high the affinity is low PREPARED BY: BATCH 2028 1D 2 BIOCHEMISTRY LC6: GLUCONEOGENESIS AND PENTOSE PATHWAY DR. ESPIRITU, A. DATE: 09/19/2024 D.2. Glucose Transporters D.5. Regulation by Reactants and Products Increase in reactants → activation leading to increase formation of products Increase in products → inhibition leading to decrease formation of products E. FATES OF PYRUVATE D.3. Fructose 2-6 bisphosphate Regulation Figure 3. Catabolic fates Pyruvate can be transformed depending on conditions Under anaerobic condition it be turned into Ethanol (we cannot form ethanol) or Lactate D.4. Phosphofructokinase 1 II. GLUCONEOGENESIS - Gluconeogenesis is the process of making new glucose from other organic molecules. Functions: Supply glucose dependent cells eg. RBCs since they lack mitochondria needed to undergo oxidative phosphorylation (via the electron transport chain) to produce Liver will release Glucagon which will ATP increase cAMP and make Fructose 2-6 Bridge periods of prolonged fasting: bisphosphate low which will lead to Example: Brain cannot utilize amino acids inactivation of Phosphofructokinase 1 since it cannot cross the BBB so it relies What happens to an actively working mostly on glucose for energy use muscle in your heart? - You expect the use of ATP - The activation of Fructose 2-6 bisphosphate will lead to activation of Phosphofructokinase 1 Figure 4. Low glucose level triggers gluconeogenesis PREPARED BY: BATCH 2028 1D 3 BIOCHEMISTRY LC6: GLUCONEOGENESIS AND PENTOSE PATHWAY DR. ESPIRITU, A. DATE: 09/19/2024 A. Precursors for Gluconeogenesis Figure 5. Comparison of Glycolysis and Gluconeogenesis. Both are thermodynamically favorable but operate in opposite directions 1. Sugars: pyruvate, lactate, oxaloacetate where the end-product is the precursor for another pathway. 2. Proteins: amino acids that can be converted to citric acid cycle intermediates Parameter Glycolysis Gluconeogenesis called, glucogenic amino acids Note: All but two amino acids (leucine and Location muscle and mainly in liver lysine) are glucogenic. brain (cytosol) (mitochondria and cytosol) Why can’t we use our fatty acid stores as alternative sources of fuel? Precursor glucose oxaloacetate Animals cannot produce glucose from fatty acid stores since its only by-product is End - product pyruvate/ glucose oxaloacetate acetyl-coA through beta-oxidation There is no net conversion of acetyl-CoA Generation of ATP is no ATP generated to oxaloacetate (precursor of ATP generated gluconeogenesis) Only applicable in plants, yeast, bacteria Steps Enzyme used Why can’t we reverse glycolysis to remake glucose 1.Pyruvate to Pyruvate Pyruvate that was broken down? oxaloacetate kinase carboxylase (PC) and Three pathways in glycolysis are PEP-carboxykinas irreversible in vivo and cannot be used in 2.Fructose 1, e (PEPCK) gluconeogenesis 6- bisphosphate Phosphofruct Fructose 1, B. Glycolysis vs. Gluconeogenesis to fructose 6 - okinase-1 6-bisphosphate phosphate 3. Glucose 6-phosphate to glucose Glucokinase/ Glucose hexokinase 6-phosphate C. Gluconeogenesis Pathway Gluconeogenesis is expensive. Energy 4 ATP, 2 GTP (guanosine (cost) triphosphate), 2 NADH Products 1 glucose molecule, 4 ADP, 2 GDP, 6 Pi (phosphate), 2 NAD+ Process Anabolic: pyruvate is a 3-carbon sugar that forms glucose, a 6-carbon sugar Physiologic Nervous system, brain, RBCs necessity generate ATP only from glucose Depletion During starvation, vigorous exercise Source/s Amino acids but not fatty acids PREPARED BY: BATCH 2028 1D 4 BIOCHEMISTRY LC6: GLUCONEOGENESIS AND PENTOSE PATHWAY DR. ESPIRITU, A. DATE: 09/19/2024 Overview of Gluconeogenesis 2. Formation of fructose 6-phosphate Precursor Enzyme Product Transporter Fructose Fructose 1, Fructose N/A 1,6-bisphosp 6-bisphosph 6-phosph Three Key Steps: hate atase ate 1. Formation of phosphoenolpyruvate + Mg2+ (2-Steps) Key notes by Doc Espiritu: Fructose 1, 6-bisphosphatase is different from fructose 2,6-bisphosphate Fructose 2,6-bisphosphate is a regulatory molecule of PFK1 and fructose 1,6-bisphosphatase that in turn regulates gluconeogenesis PFK2 is bifunctional which has phosphofructokinase-2 and phosphofructobisphosphatase domains Precursor Enzyme Product Transporter 3. Formation of the end-product, 1 1. Pyruvate Pyruvate Oxaloacet Malate molecule of glucose carboxylase ate aspartate + (2) ATP (synthesiz shuttle ( ed in mitochond mitochon ria to the dria then cytosol) converted to malate for transport) 2. Phosphoen Phosphoe N/A Oxaloacetat olpyruvate nolpyruva e (converted carboxykina te (PEP) from malate se (PEPCK) by malate + Mg2+ dehydrogen ase) Precursor Enzyme Product Transporter Fructose Glucose-6-p Glucose Glucose-6- Important characteristics of pyruvate carboxylase: 6-phosphate hosphatase phosphate + Mg2+ translocase Biotin dependent mitochondrial enzyme (cytosol to ER) that converts pyruvate to oxaloacetate in Keynote by Doc Espiritu: presence of ATP & CO2 Glucose 6-phosphate is catalyzed by Regulates gluconeogenesis glucose 6-phosphatase (only found in Requires acetyl CoA) endoplasmic reticulum) into glucose Key notes by Doc Espiritu: which can be released to the blood Oxaloacetate is also a precursor of Important characteristics of glucose 6-phosphatase: tricarboxylic acid cycle (TCA) together with Present in liver and kidney but absent in acetyl-CoA. The formation of oxaloacetate muscle, brain, adipose tissue to enter the TCA cycle is called Needs magnesium as cofactor Anaphlerotic reaction. Energy balance: Since it is an anabolic process, *The next steps are similar to glycolysis as well as there is a need for energy, we need: the enzymes used until phosphoenolpyruvate (PEP) becomes fructose 1, 6-bisphosphate. These are A total of 4 ATP is needed, 2 GTP and 2 NADH and also reversible. H+ for the formation of 1 glucose molecule from 2 pyruvate molecules PREPARED BY: BATCH 2028 1D 5 BIOCHEMISTRY LC6: GLUCONEOGENESIS AND PENTOSE PATHWAY DR. ESPIRITU, A. DATE: 09/19/2024 D. Gluconeogenesis Regulator and D.1. Glucogenic Amino Acids Key processes Glucogenic amino acids = able to undergo net conversion to glucose Intermediates of the citric acid cycle can also undergo oxidation to oxaloacetate F. Gluconeogenesis From Glycerol Acetyl-CoA (substrate of citric acid cycle) Glycerol is liberated in the adipose tissue is an activator of pyruvate carboxylase by the hydrolysis of fats (triacylglycerols). ADP is an inhibitor of both the pyruvate The enzyme glycerokinase (found in liver carboxylase and phosphoenolpyruvate and kidney, absent in adipose tissue) carboxykinase activates glycerol to glycerol 3-phosphate. It is converted to DHAP by glycerol 3-phosphate dehydrogenase. DHAP is an intermediate in glycolysis. G. Gluconeogenesis From Propionate Oxidation of odd chain fatty acids & the breakdown of some amino acids *Reciprocal Regulation (methionine, isoleucine) yields a three carbon propionyl COA. Fructose-1,6-bisphosphatase is activated Propionyl COA carboxylase acts on this in by citrate, and inhibited by AMP, and the presence of ATP & biotin & converts to Fructose-2,6-bisphosphate methyl melony! COA Opposite reaction of the Which is then converted to succinyl CoA in Fructose-2,6-bisphosphate (activating the the presence of B12 forward reaction in glycolysis, and Succinyl CoA formed from propionyl CoA inhibition action in the gluconeogenesis) enters gluconeogenesis. reciprocal reaction: regulation by the same compound H. Gluconeogenesis From Lactate reciprocal regulation: opposite effect (Cori Cycle) E. Gluconeogenesis From Amino It is a process in which glucose is Acids converted to Lactate in the muscle and in the liver this lactate is re-converted to The carbon skeleton of glucogenic amino glucose. acids (all except leucine & lysine) results in In an actively contracting muscle, pyruvate the formation of pyruvate or the is reduced to lactic acid which may tend to intermediates of citric acid cycle. accumulate in the muscle. Which, ultimately, results in the synthesis To prevent lactate accumulation, body of glucose. utilizes cori cycle (Anaerobic respiration) Ketogenic amino acid: Converted from carbon skeleton of fatty acids PREPARED BY: BATCH 2028 1D 6 BIOCHEMISTRY LC6: GLUCONEOGENESIS AND PENTOSE PATHWAY DR. ESPIRITU, A. DATE: 09/19/2024 III. ANAEROBIC RESPIRATION The lactate can be transported to the liver and converted to glucose there. Requires a recovery time ○ high amount of oxygen consumption to fuel gluconeogenesis ○ restores muscle glycogen store A. Anaerobic Glycolysis: Fermentation In glycolysis, dehydrogenation of the two molecules of glyceraldehyde 3-phosphate derived from each Generation of energy (ATP) without molecule of glucose converts two molecules of consuming oxygen or NAD+ NAD+ to two of NADH. Because the reduction of No net change in oxidation state of the two molecules of pyruvate to two of lactate sugars regenerates two molecules of NAD+, there is no net Reduction of pyruvate to another product change in NAD+ or NADH. Regenerates NAD+ for further glycolysis under anaerobic conditions The process is used in the production of food from beer to yogurt to soy sauce. Under hypoxic conditions, however, NADH generated by glycolysis cannot be reoxidized by 02. Failure to regenerate NAD+ would leave the cell with no electron acceptor for the oxidation of glyceraldehyde 3-phosphate, and the Lactate dehydrogenase is an important energy-yielding reactions of glycolysis enzyme. It can be used as a cardiac would stop. NAD+ must therefore be biomarker for myocardial infarction. It has regenerated in some other way. different isoforms: ○ LDH-1 specific to cardiac muscle; B. Lactic Acid Fermentation Advantage to Troponin Amino acid undergo Lactic acid ○ LDH-2 fermentation ○ LDH-3 When animal tissues cannot be supplied ○ LDH-4 with sufficient O2 to support aerobic ○ LDH-5 oxidation of the pyruvate and NADH produced in glycolysis, NAD+ is regenerated from NADH by the reduction of pyruvate to lactate Reduction of pyruvate to lactate via the lactate dehydrogenase ○ reversible reaction ○ Lactate is the final product of anaerobic glycolysis During strenuous exercise, lactate builds up in the muscle. - generally, less than 1 minute The acidification of muscle prevents its continuous strenuous work. PREPARED BY: BATCH 2028 1D 7 BIOCHEMISTRY LC6: GLUCONEOGENESIS AND PENTOSE PATHWAY DR. ESPIRITU, A. DATE: 09/19/2024 C. The Cori Cycle B. Two Phases of the Pentose Phosphate Pathway AKA Hexose Monophosphate Shunt Occurs in all cell types but primarily in the liver - Generally, 10% of glucose in metabolized thru the PPP - In the liver is 30% - Oxidation of glucose to form ribose-5-phosphate, the precursor of nucleic acid - 2 phases: oxidative and non-oxidative The Cori Cycle Lactate is used as a precursor in the gluconeogenesis Bloodborne glucose is converted by exercising muscle to lactate, which C. Pentose Phosphate Pathway diffuses into the blood. This lactate is taken up by the liver and reconverted to glucose, through The main function is to produce NADPH Gluconeogenesis, which is released back and ribose 5-phosphate. into the circulation and the cycle continues NADPH is an electron donor. The glucose that is formed in the liver can - reductive biosynthesis of fatty acids now be transferred back into the actively and steroids (50%) exercising muscle. - repair of oxidative damage (Reducing molecule against oxidative stress) IV. PENTOSE PHOSPHATE PATHWAY - Although oxygen is important in cellular processes, too much oxygen A. Four Major Pathways of Glucose can form reactive oxygen species Utilization which oxidizes the cell. That is why NADPH is needed to reduce this reactive oxygen. - Important in cytochrome P450 enzyme Ribose-5-phosphate is a biosynthetic precursor of nucleotides. - used in DNA and RNA synthesis or synthesis of some coenzymes Also interconverts sugars - to produce trioses, hexoses and pentoses PREPARED BY: BATCH 2028 1D 8 BIOCHEMISTRY LC6: GLUCONEOGENESIS AND PENTOSE PATHWAY DR. ESPIRITU, A. DATE: 09/19/2024 - There are 2 main functional Ribulose-5-phosphate can be used in two groups aside from hydroxyl: pathways: aldose and ketoses Ribulose-5-phosphate can be used to generate - Transketolase - transfer of ketose ribose-5-phosphate (thru ribose - phosphate sugar hydrolase) for synthesis of DNA/RNA - Transaldoses - transfer of aldose (nucleotides. sugar Ribulose-5-phosphate can also be used to generate xylulose-5-phosphate (thru epimerase) for non-oxidative PPP and can be further converted to fructose-6-phosphate. The fructose-6-phosphate is the intermediate product of glycolysis, so this can go back (shunted back) to glycolysis. C.1. Oxidative Phase Generates NADPH and a Pentose - Glucose will be taken up by the cell and will be enacted by hexokinase or glucokinase which converts it to glucose-6-phosphate. Some of the glucose-6-phosphate will undergo glycolysis and 10% generally or 30% in the liver will undergo PPP. - In PPP, the glucose 6-phosphate will be acted by glucose-6-phosphate dehydrogenase with the use of NADP+ and magnesium to produce 6-phospho-glucono-lactone. D. NADPH and Oxidative Stress - The 6-phospho-glucono-lactone will be - The formation of NADPH will reduce acted by lactonase to produce glutathione using glutathione reductase 6-phospho-gluconate. enzyme to form reduced glutathione. - 6-phospho-gluconate will be converted to - The reduced glutathione can reduce reactive ribulose-5-phosphate thru the oxygen species (e.g. hydrogen peroxide that is 6-phospho-gluconate dehydrogenase. toxic to cell) with the help of glutathione - End product of the oxidative phase: peroxidase. This breaks down hydrogen ribulose-5-phosphate and production of peroxide to 2 molecules of water NADPH. PREPARED BY: BATCH 2028 1D 9 BIOCHEMISTRY LC6: GLUCONEOGENESIS AND PENTOSE PATHWAY DR. ESPIRITU, A. DATE: 09/19/2024 E. Nonoxidative Phase Regenerates G. Summary G-6-P from R-5-P Used in tissues requiring more NADPH Glycolysis, a process by which cells can than Ribose-5-Phosphate extract a limited amount of energy from - Such as the liver and adipose glucose tissue Fermentation, a process by which cells The ribose-5-phosphate will be shunted to can continue using glycolysis to extract the formation of glucose-6-phosphate energy in anaerobic conditions which can enter glycolysis. Gluconeogenesis, a process by which cells can use a variety of metabolites for the synthesis of glucose The differences between glycolysis and gluconeogenesis - how they are both made thermodynamically favorable - how they are differentially regulated to avoid a futile cycle The pentose phosphate pathway, a process by which cells can generate pentose phosphates and NADPH. The pentose phosphates can be regenerated into glucose-6-phosphate, which requires no ATP. F. PPP Clinical Correlates: Glucose-6-Phosphate Dehydrogenase Deficiency Deficiency of G6PD Reference(s): - 7.5% of world population deficient 1. Dr. A. Espiritu (2024). Lecture and Powerpoint - 35% prevalence in certain areas Presentation. of Africa 2. Lehninger Principles of Biochemistry (6th ed.). W. H. Freeman and Company - X-linked recessive inheritance - Most common inborn errors of metabolism - Leads to inadequate formation of NADPH that combat oxidative stress May cause: - Hemolytic Anemia (oxidative stress targets RBC because it is where the concentration of oxygen is high) Oxidative triggers: - Infections: Typhoid fever, pneumonia - Drugs: Antimalarial, sulfonamides, nitrofurantoin analgesics - Certain food: fava beans (favism) PREPARED BY: BATCH 2028 1D 10