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Glucose Metabolism Pratt and Cornely, Chapter 13 Glycolysis Expectations • • • • Memorize/learn Figure 13.2 Know overall reaction and stages Explain chemical logic of each step Enzyme mechanisms presented in book Glycolysis • Ten enzymes that take glucose to pyruvate • Cytosol • ATP and NADH Reactions and Enzymes of Glycolysis ATP ATP Pi + NAD+ ADP 2x ADP ADP NADH ADP 2x 2x ATP 2x ATP • Hexose and triose phases • Energy input and payoff phases Energy Input Energy Payoff Know... • • • • Substrates Co-substrates Products Enzyme names 1. Hexokinase • Previous concepts: Induced fit, kinase • Utilizes 1 ATP • Chemical logic? Problem 3 • (Notice miswording) The DGo’ value for hexokinase is -16.7 kJ/mol, but it is twice as spontaneous under cellular conditions. – What is the ratio of G-6-P to glucose under cellular conditions when the ratio of ATP:ADP is 10:1? – How high would the ratio of G-6-P to glucose have to be to reverse the hexokinase reaction by mass action? 2. Phosphoglucose Isomerase • • • • Previous concepts: Isomerization CONCEPT: Near-equilibrium Chemical logic: Stereochemistry—reverse does not produce mannose! 3. PFK-1 • • • • Previous concepts: Allosteric inhibition Utilizes 1 ATP Pathway logic: First committed step of glycolysis – Why? – regulation Regulation: Bacteria vs. Human 4. Aldolase • Previous concepts: Standard free energy is +23kJ, but it is a near equilibrium reaction • Pathway logic: • Beginning of triose stage Aldolase Mechanism 5. Triose Phosphate Isomerase • Previous concepts: Catalytic perfection • Pathway logic: • Most similar to which previous reaction? 6. Glyceraldehyde-3-P DH • Previous concepts: Redox and dehydrogenase • Pathway logic: Utilizes negative free energy of _____________ to drive nonspontaneous formation of ___________________ GAPDH Mechanism Be able to draw mechanism with full NAD+ structure 7. Phosphoglycerate Kinase • • • • Previous concepts: High energy bond Produces 2 ATP: substrate level phosphorylation Pathway logic? Coupled to reaction 6 by Le Chatelier Coupled Reactions • GAPDH = 6.7 kJ/mol • PG Kinase = -18.8 kJ/mol • Overall: A set of near-equilibrium reactions at cellular concentration 8. Phosphoglycerate Mutase • Previous concepts: Covalent catalysis • Pathway logic: • Mutase—isomerization with P transfer Mechanism • Not a simple transfer • What happens if the bisphosphate escapes? 9. Enolase • Concept: Phosphoryl group transfer potential • Chemical logic? 10. Pyruvate Kinase • Production of 2 ATP • VERY high energy bond allows formation of _________ while still being irreversible • Regulation: F-1,6-BP can act as a feed-forward activator to ensure fast glycolysis Overall Energetics • Standard Free energies are up and down • Free energies under cellular conditions are downhill or near zero – Three irreversible Fate of Pyruvate Amino acid and nitrogen metabolism Gluconeogenesis Aerobic Energy Anaerobic in higher organisms Anaerobic in microorganisms The Problem of Anaerobic Metabolism • With oxygen, the NADH produced in glycolysis is re-oxidized back to NAD+ • NAD+/NADH is a co-substrate which means… • If there is no oxygen, glycolysis will stop because… • The solution to the problem is to… The solution in Yeast • Pyruvate is decarboxylated to acetaldehyde • Acetaldehyde transformed to ethanol – What type of reaction? – What cofactor? • NAD+ is regenerated to be reused in GAPDH The Solution in Us • Lactate formation: Draw a mechanism • Balanced equation We don’t operate anaerobically... • Most energy still trapped in lactate • Back to pyruvate, then acetyl-CoA • Citric acid cycle Other sugars enter glycolysis High fructose diet puts sugars through glycolysis while avoiding major regulation step Glucose Metabolism Overview • Keep the main pathway purposes distinct • But learn details of chemistry and regulation based on similarities Glucose Metabolism Overview • Gluconeogenesis • Glycogen metabolism • Pentose Phosphate Pathway Precursors for Gluconeogenesis • Names of compounds? • Type of reaction? • Type of enzyme? • Cofactor(s)? Chemistry of Gluconeogenesis • Pyruvate transformed to glucose • But energetically costly—no perpetual motion machine! • Points of regulation Glycolysis • Gluconeogenesis • • • • • Step 10: no ATP back • Step 8: no ATP back • Step 3: costs 2 ATP • Step 1a/b: costs 4 ATP equivalents Step 1: costs 1 ATP Step 3: costs 1 ATP Step 7: makes 2 ATP Step 10: makes 2 ATP Step 1a • Pyruvate Carboxylase – Biotin – Costs ATP to make driving force for next reaction • TIMES TWO! – First step in biosynthesis of glucose and many other molecules • Related to which amino acid? Mechanism • Mixed anhydride activates carboxyl group • Coupled through biotin coenzyme • Enolate attacks activated carboxylate Step 1b • PEP carboxykinase – ATP cost to restore PEP • TIMES TWO! – CO2 loss drives rxn Step 8 • Fructose-1,6-bisphosphatase • No additional energy input • Phosphate ester hydrolysis is spontaneous Step 10 • Glucose 6-phosphatase – Liver (and others) – Not in muscle Problem 34 • A liver biopsy of a four-year old boy indicated that the F-1,6-Bpase enzyme activity was 20% normal. The patient’s blood glucose levels were normal at the beginning of a fast, but then decreased suddenly. Pyruvate and alanine concentrations were also elevated, as was the glyceraldehyde/DHAP ratio. Explain the reason for these symptoms. Key Regulation • At the committed step in glucogenic cells • Principle of Reciprocal Regulation • Local regulation vs Hormone regulation Key Regulation • Local regulation – AMP/ATP (energy charge) – Citrate (feedback) • Hormone regulation – Fructose-2,6-bisphosphate • Gluconeogenesis is inhibited • Glycolysis is stimulated Problem 39 • Brazilin, a compound found in aqueous extracts of sappan wood, has been used to treat diabetics in Korea. It increases the activity of the enzyme that products F-2,6-BP and stimulates the activity of pyruvate kinase. What is the effect of adding brazilin to liver cells in culture? Why would brazilin be an effective treatment for diabetes? Glucose Metabolism Overview • Gluconeogenesis • Glycogen metabolism • Pentose Phosphate Pathway Glycogen • • • • Storage molecule Primer necessary Very large! Multiple ends allow for quick synthesis and degradation Chemistry of Synthesis • Step 1 • Near equilibrium • The link to glucose-6-phophate, our central molecule Chemistry of Synthesis • Step 2 • Count high energy bonds • Pyrophosphatase – Common motiff • UDP-glucose: activated for incorporation Chemistry of Synthesis • Step 3 • Glycogen synthase • Growing end is non-reducing • UDP released Branching Energetics of Synthesis • Total cost: one ATP equivalent from G-6-P Four Degradation Enzymes • Two enzyme debranching process • Glycogen phosphorylase • phophoglucomutase Chemistry of Degradation • Glycogen phosphorylase • Key Regulation site • Inorganic phosphate as a nucleophile • Remake G-1-P with no ATP cost Debranching • Transfer branch • a(16)glycosidase Overall Energetics Key Regulatory Enzymes Glycogen Storage Diseases Many disrupt glycogen breakdown in muscle and/or liver (hypoglycemia, enlarged liver, muscle cramps...) Glucose Metabolism Overview • Gluconeogenesis • Glycogen metabolism • Pentose Phosphate Pathway Pentose Phosphate Pathway • Dual Purpose – Synthesis of “reducing potential” – Synthesis of 5-carbon sugars • At cost of one carbon worth of carbohydrate • Net reaction: Physiological Purposes Glutathione: Protection from Oxidation • Glutathione is the redox buffer of the cell • Regenerated by NADPH • PPP especially important in RBC because it is only means to generate reducing power (no mitochondria) Complex, 2-Stage Process • Oxidative Stage – Generates reducing power and ribose • Non-oxidative stage – Regenerates 3- and 6-carbon sugars from 5 carbon sugars Oxidative Stage Step 1: • G-6-P DH • Lactone formation Oxidative Stage Step 2: • Also a spontaneous hydrolysis • Practice mechanism, carbohydrate orientation Oxidative Stage Step 3: • Oxidative decarboxylation – Which is first, oxidation or decarboxylation? • We will see this process again Biosynthesis of Ribose Non-oxidative Stage • To understand purpose, realize that we generally need to make much more NADPH than ribose • Problem: stuck with C5, but need C6 and C3 • Solution: “Shunt” C5 back to C6 through near-equilibrium reactions PPP Reactions • • • • Epimerase Isomerase Transketolase Transaldolase Epimerase Transketolase • Use cofactor (B1) to overcome chemical problem Mechanism Transketalase, Transaldolase • Transketalase – Transfer of an acyl anion – Requires TPP to stabilize unstable anion • Transaldolase – Transfer of somewhat stable enolate ion – Schiff base helps • Be able to recognize need for TK vs. TA Transketalase or transaldolase? Different Modes for Different Purposes Problem 58 • A given metabolite may follow more than one metabolic pathway. List all possible fates of glucose-6-P in (a) a liver cell and (b) a muscle cell. Summary of glucose metabolism