Glycolysis: Energy Generation Without an Oxygen Requirement Glucose Biofuel Prominence: Low-reactive ring-form minimizes protein glycosylation

Glycolysis: A Three Step Process Glucose trapping and destabilization (priming) Three carbon unit generation (cleaving) Energy generation

Induced Fit in Hexokinase Glucose induces a large enzyme conformational change Substrate-induced cleft closing prevents ATP hydrolysis Kinases require a divalent metal ion What function does Mg+2 play in hexokinase?

Hexokinase Closed Around Substrates What mechanisms of catalysis are operative?

Hexokinase Reaction Mechanism What is the Nu:, electrophile, and leaving group in this reaction?

Phosphoglucose Isomerase: Aldose to Ketose Conversion

PGI Reaction Mechanism Phosphoglucose Isomerase (PGI) G6P Conversion via Acid-Base Catalysis PGI Reaction Mechanism

Phosphoglucose Isomerase (PGI) G6P Conversion via Acid-Base Catalysis Base catalyzed bond formation

Phosphoglucose Isomerase (PGI) G6P Conversion via Acid-Base Catalysis Acid catalyzed ketal formation

Base catalyzes ring closure Phosphoglucose Isomerase (PGI) G6P Conversion via Acid-Base Catalysis Base catalyzes ring closure H+

Phosphofructokinase: Trapping the Fructose Isomer What is the mechanism for this reaction?

Glycolysis Stage I: Glucose Trapping and Destabilization (priming)

Six Carbon Sugar Cleaved to Two Three Carbon Units What is the bond to be cleaved? Which alcohol becomes an aldehyde?

Haworth and Fischer Projections Equivalency The functional group that is down in a Haworth projection is positioned how in a Fischer structure?

Aldolase Reaction Mechanism Fructose-1,6-bisphosphate binds to the aldolase enzyme for covalent catalysis

Aldolase Reaction Mechanism What is lost when the Schiff base forms?

Aldolase Rxn Mechanism Aldolase Reaction Mechanism Aldolase Rxn Mechanism Compare and contrast a Schiff base with a carbonyl group.

Aldolase Reaction Mechanism What is the process for Schiff base to carbonyl conversion?

Aldolase Reaction Mechanism H2O Aldolase cleaves FBP into GAP and DHAP

Triose Phosphate Isomerase (TIM) Reversible and driven towards GAP due to product depletion Which previous glycolytic step is similar to TIM?

Glycolysis: Step #5 Triose Phosphate Isomerase Triose Phosphate Isomerase Reaction Mechanism Glycolysis: Step #5 Triose Phosphate Isomerase TIM- or α,β-barrel with 8 parallel β-strands surrounded by 8 α-helices. DHAP conversion to GAP necessary to proceed through glycolysis

Stoichiometry: Stages 1-2 of Glycolysis Two ATPs are initially invested. One glucose is metabolized into two GAP molecules.

Road Map for Energy Harvest (Stage 3)

Glyceraldehyde-3-Phosphate Dehydrogenase: Covalent Catalysis

Glyceraldehyde-3-Phosphate Dehydrogenase: a 2 Step Process What amino acid will serve as a nucleophile to form a thioester?

Glyceraldehyde-3-Phosphate Dehydrogenase: Reaction Mechanism

Actual coupled reaction Glyceraldehyde-3-Phosphate Dehydrogenase: Catalysis Energetics Hypothetical reaction with no coupling Actual coupled reaction

Phosphoglycerate Kinase What is the Nu:, electrophile and leaving group for this reaction? (hint: consider hexokinase in reverse)

Glycolysis: the Three Final Steps

Pyruvate Kinase What is the Nu:, electrophile and leaving group for this reaction? (hint: consider phosphoglycerate kinase)

Glycolysis Energetic ∆G°ʹ ∆G Enzyme (kcal/mol) (kcal/mol) 1near equilibrium means that ∆G is about zero What is the relationship between ∆G and ∆G°ʹ? When can ∆G and ∆G°ʹ diverge?

Regulating Glycolysis: A Pictorial Analogue Water represents metabolite flux Water amount in flask represents intermediate abundance Flasks connections are enzymes Vertical drop represents decrease in free energy ΔG° = height difference between flask bottoms ΔG = height difference between water levels

Metabolic Regulation Irreversible reactions are potential regulatory sites (e.g. hexokinase, phosphofructokinase and pyruvate kinase) What duel role does ATP play in PFK-1 catalysis? In what direction does ATP regulate phosphofructokinase?

Energy Status Regulates Glycolytic Flow Elevated [ATP] sufficient energy; elevate [AMP] low energy ADP + ADP ↔ ATP + AMP <adenylate kinase> Muscle Tissue

Fructose-2,6-Bisphosphate an Allosteric Regulator of Phosphofructokinase-1 PFK-2 Liver Tissue Front activation by fructose-6P F-2,6-BP amplifies or diminishes PFK-1 activity?

Fructose-2,6-Bisphosphate Reduces ATP Inhibition of Phosphofructokinase-1 PFK-2 Liver Tissue ATP is a substrate and inhibitor of PFK-1

Fructose Entry Points for Glycolysis Glycerol-3P Glucose + Fructose Major dietary sugars: sucrose (table sugar) and fructose (high-fructose corn syrup)

Fructose Metabolism How is this different than glucose metabolism?

Fructose Metabolism Glycerol 3-phosphate a precursor to triacylglycerol Fructose catabolism bypasses phosphofructokinase regulation Glycerol 3-Phosphate Lipid Synthesis

Alternative Fates for Pyruvate

Anaerobic Recycling of NADH for Glycolysis

Microbial Recycling of NADH for Glycolysis

Pyruvate Dehydrogenase: the Bridge between Glycolysis and Citric Acid Cycle

Standard Free Energy Change Comparisons for Glucose Catabolism With and Without Oxygen

Pathogenic Obligate Anaerobes

Pyruvate Targeted for Anabolism The biotin prosthetic group serves as a CO2 carrier What reaction links biotin to the protein?

Pyruvate Carboxylase: an Endergonic Reaction Oxaloacetate

Glucose Metabolism: Both Catabolic and Anabolic

Glucose Metabolism: Both Catabolic and Anabolic

Problems: 1, 3, 5, 7, 13, and 21