Step 8: Migration of the Phosphate Step 8: Migration of the Phosphate • Rationale: – Be able to form high-energy phosphate compound • Mutases catalyze the (apparent) migration of functional groups – One of the active site histidines is post-translationally modified to phosphohistidine – Phosphohistidine donates its phosphate to O2 before retrieving another phosphate from O3 • 2,3-bisphosphoglycerate intermediate • Note that the phosphate from the substrate ends up bound to the enzyme at the end of the reaction Step 8: Migration of the Phosphate • Thermodynamically unfavorable/reversible – Reactant concentration kept high by PGK to push forward Mechanism of Phosphoglycerate Mutase: Base Catalyzed Phosphoryl Transfer Mechanism of Phosphoglycerate Mutase: Acid Catalyzed Phosphoryl Transfer Step 9: Dehydration of 2-PG to PEP Step 9: Dehydration of 2-PG to PEP • Rationale – Generate a high-energy phosphate compound • 2-Phosphoglycerate is not a good enough phosphate donor – Two negative charges in 2-PG are fairly close – But loss of phosphate from 2-PG would give a secondary alcohol with no further stabilization • Slightly thermodynamically unfavorable/reversible – Product concentration kept low to pull forward Step 10: 2nd Production of ATP Step 10: 2nd Production of ATP • Rationale – Substrate-level phosphorylation to make ATP – Net production of 2 ATP/glucose • Loss of phosphate from PEP yields an enol that tautomerizes into ketone – Tautomerization • effectively lowers the concentration of the reaction product • drives the reaction toward ATP formation Pyruvate Tautomerization Drives ATP Production Step 10: 2nd Production of ATP • Pyruvate kinase requires divalent metals (Mg2+ or Mn2+) for activity • Highly thermodynamically favorable/irreversible – Regulated by ATP, divalent metals, and other metabolites 4C. Summary of Glycolysis Glucose + 2 NAD+ + 2 ADP + 2 Pi 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP • Used: – 1 glucose; 2 ATP; 2 NAD+ • Made: – 2 pyruvate • Various different fates – 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 – Ensure proper use of nutrients – Ensure production of ATP only when needed 4D. Fates of Pyruvate 5. Glycolysis occurs at elevated rates in tumor cells Tumor cells have a higher requirement for glucose due to a lower efficiency in energy production from glycolysis. • Complete oxidation of CO2 in healthy cells under aerobic conditions yields ~30 ATP per glucose. • Anaerobic metabolism of glucose in tumor cells yields 2 ATP per glucose. – Glucose transporters and most glycolytic enzymes are overexpressed in tumors versus normal cells. – Inhibitors of glycolytic pathways could be effective anticancer drugs. Gluconeogenesis 15 1. The Body’s Glucose Need In mammals, some tissues depend almost completely on glucose for their metabolic energy. Glucose from the blood is the sole or major fuel source for: • Human brain and nervous system - Brain requires 120 g/day, more than half that is stored as glycogen in muscles and liver. • Erythrocytes • Testes • Renal medulla • Embryonic tissues A mechanism for our bodies to produce glucose is crucial. 2. Gluconeogenesis: Precursors for Carbohydrates Notice that mammals cannot convert fatty acids to sugars. 2. Gluconeogenesis: Precursors for Carbohydrates • Animals can produce glucose from sugars or proteins – Sugars: pyruvate, lactate, or oxaloacetate – Protein: from amino acids that can be converted to citric acid cycle intermediates (or glucogenic amino acids) • Animals cannot produce glucose from fatty acids – Product of fatty acid degradation is acetyl-CoA – Cannot have a net conversion of acetyl-CoA to oxaloacetate • Plants, yeast, and many bacteria can do this, thus producing glucose from fatty acids 3. Glycolysis vs. Gluconeogenesis Glycolysis occurs mainly in the muscle and brain. Gluconeogenesis occurs mainly in the liver. 3. Glycolysis vs. Gluconeogenesis • Opposing pathways that are both thermodynamically favorable – Operate in opposite direction • end product of one is the starting compound of the other • Reversible reactions are used by both pathways • Irreversible reaction of glycolysis must be bypassed in gluconeogenesis – Highly thermodynamically favorable, and regulated – Different enzymes in the different pathways – Differentially regulated to prevent a futile cycle 4. Three bypass reactions of gluconeogenesis. A. Conversion of pyruvate to phosphoenolpyruvate B. Conversion of fructose 1,6-bisphosphate to fructose-6phosphate C. Conversion of glucose-6-phosphate to glucose These bypasses are irreversible steps. 4A. Pyruvate to Phosphoenolpyruvate • Requires two energy-consuming steps • First step, pyruvate carboxylase converts pyruvate to oxaloacetate – Carboxylation using a biotin cofactor – Requires transport out of the mitochondria via malate • Second step, phosphoenolpyruvate carboxykinase converts oxaloacetate to phosphoenolpyruvate – Phosphorylation from GTP and decarboxylation – Occurs in mitochondria or cytosol depending on the organism 4AIa. Synthesis of Oxaloacetate This reaction occurs in the mitochondria. Biotin in a cofactor of the enzyme. 4AIb. Biotin is a CO2 Carrier 4AIc. Oxaloacetate conversion to malate Oxaloacetate has to be converted to malate for it to be transported out of the mitochondrion. Mitochondrion Cytosol 4AIIa. Oxaloacetate to Phosphoenolpyruvate Summary of first bypass reaction. Pyruvate + ATP + GTP + HCO3- → PEP + ADP + GDP + Pi + CO2 ΔG’°= 0.9 kJ/mol The reaction is irreversible due to the ready consumption of PEP, decreasing the amount of product. 4AIII. From Pyruvate to Phosphoenolpyruvate Isozymes: Two distinct enzymes that catalyze the same reaction. They can have different cellular locations or metabolic roles. 4B. Second bypass reaction Fructose-1-6bisphosphatase + Mg2+ + H2O + Pi ΔG’°= -16.3 kJ/mol 4C. Thirds bypass reaction Glucose-6-phosphatase Glucose-6-phosphate + Mg2+ + H2O Glucose + Pi ΔG’°= -13.8 kJ/mol 5. Gluconeogenesis is expensive 2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H+ + 4 H2O Glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+ • Costs 4 ATP, 2 GTP, and 2 NADH but physiologically necessary – Brain, nervous system, and red blood cells generate ATP ONLY from glucose Pentose Phosphate Pathway 33 1. Pentose Phosphate Pathway 1. Pentose Phosphate Pathway • The main products are NADPH and ribose 5-phosphate • NADPH is an electron donor – Reductive biosynthesis of fatty acids and steroids – Repair of oxidative damage • For certain tissue, a reducing atmosphere (high ratio of NADPH to NADP+ and a high ratio of reduced to oxidized glutathione) helps combat damage by reactive oxygen species. • Ribose-5-phosphate is a biosynthetic precursor of nucleotides – Used in DNA and RNA synthesis – Or synthesis of some coenzymes 2. NADPH regulates partitioning into glycolysis vs. pentose phosphate pathway