Biochem - 9.3 notes (book + lecture)

Q: What does Figure 9.15 show?

A: Figure 9.15 shows glycolysis at the top of a set of interconnected pathways that include the citrate cycle and oxidative phosphorylation. Together, these pathways completely oxidize glucose to CO₂ and H₂O.

Q: What is the overall oxidation reaction of glucose?

A:
Glucose (C₆H₁₂O₆) + 6 O₂ ⇌ 6 CO₂ + 6 H₂O
ΔG°′ = −2867 kJ/mol
ΔG = −2938 kJ/mol

Q: Why is glycolysis considered a core metabolic pathway?

A:

1. Glycolytic enzymes are highly conserved among all living organisms → ancient pathway.

2. Primary pathway for ATP generation under anaerobic conditions and in cells lacking mitochondria (e.g., erythrocytes).

3. Glycolytic metabolites are precursors for many interdependent pathways, including mitochondrial ATP synthesis.

Q: (Example) How was glycolysis discovered?

A: It was elucidated in the early 1900s by chemists studying fermentation in brewer’s yeast, following Eduard Buchner’s experiments showing that yeast cell extracts contained everything needed for fermentation in a test tube.

Q: What does glycolysis accomplish for the cell?

A: It generates a small amount of ATP under anaerobic conditions and produces pyruvate, the precursor to acetyl-CoA, lactate, or ethanol.

Q: What is the overall net reaction of glycolysis?

Glucose + 2 NAD⁺ + 2 ADP + 2 Pi ⇌ 2 Pyruvate + 2 NADH + 2 H⁺ + 2 ATP + 2 H₂O

Q: What are the key enzymes in glycolysis?

A:

• Hexokinase: Catalyzes the first step; inhibited by glucose-6-phosphate.

• Phosphofructokinase-1 (PFK-1): Allosterically activated by AMP and fructose-2,6-bisphosphate; inhibited by ATP and citrate.

• Pyruvate kinase: Catalyzes the final step; activated by AMP and fructose-1,6-bisphosphate; inhibited by ATP and acetyl-CoA.

Q: (Example) What is an example of glycolysis in everyday biochemistry?

A: Glycolysis regulates blood glucose. Deficiency in glucokinase (a hexokinase-related enzyme) causes maturity-onset diabetes of the young (MODY2), due to inability of liver/pancreatic cells to phosphorylate glucose → high blood glucose.

Q: How many enzymatic reactions make up glycolysis?

A: 10 enzymatic reactions.

Q: What does the glycolytic pathway accomplish?

A: Converts one glucose molecule to two pyruvates in the cytosol, producing a net gain of 2 ATP.

Q: What happens to pyruvate after glycolysis under aerobic conditions?

A: Pyruvate enters mitochondria, oxidized to CO₂ and H₂O, producing 30 more ATP — total 32 ATP per glucose.

Q: (Figure 9.16 concept) What advantage does glycolysis have under anaerobic conditions?

A: Glycolysis can generate 2 ATP without oxygen — sustaining ATP production when O₂ is limited (e.g., during exercise).

Q: What is the energy yield comparison between aerobic and anaerobic metabolism?

A: Glycolysis alone (anaerobic): 2 ATP per glucose (≈6% yield).
Complete aerobic oxidation: 32 ATP per glucose.

Q: What provides most of the ATP in organisms?

A: Oxidation of pyruvate and acetyl-CoA in the mitochondria.

Q: What is the major energy source for ATP in animals?

A: Stored fats (fatty acids) → oxidized via fatty acid oxidation and acetyl-CoA metabolism in mitochondria.

Q: What conservation occurs in glycolysis?

A: The six carbons and six oxygens from glucose are conserved in the two pyruvate molecules produced.

Q: What do the 10 glycolytic reactions involve?

A: Enzyme-catalyzed phosphoryl transfers, isomerizations, aldol cleavage, oxidation, and dehydration—without net carbon or oxygen loss.

Q: What are the two stages of glycolysis?

• Stage 1: ATP investment (reactions 1–5)

• Stage 2: ATP earnings (reactions 6–10)

Q: What is required in Stage 1 of glycolysis?

A: Chemical energy input as phosphoryl transfer generates phosphorylated compounds for Stage 2.

Q: What enzyme catalyzes the first step of glycolysis?

A: Hexokinase (in all cells) phosphorylates glucose using ATP → glucose-6-phosphate (G6P).
Glucokinase (liver/pancreas) performs the same function.

Q: What happens to glucose-6-phosphate next?

A: It is isomerized by phosphoglucose isomerase to fructose-6-phosphate (F6P).

Q: What enzyme phosphorylates F6P?

A: Phosphofructokinase-1 (PFK-1) uses ATP to form fructose-1,6-bisphosphate (F1,6-BP).

Q: What happens in reaction 4 of glycolysis?

A: Aldolase cleaves F1,6-BP into two 3-carbon products:

• Glyceraldehyde-3-phosphate (G3P)

• Dihydroxyacetone phosphate (DHAP)

Q: What happens to DHAP?

A: It is isomerized by triose phosphate isomerase into another G3P molecule.

Q: What reaction begins Stage 2 of glycolysis?

A: Oxidation of glyceraldehyde-3-phosphate by G3P dehydrogenase (GAPDH), forming 1,3-bisphosphoglycerate.

Q: What is substrate-level phosphorylation?

A: Direct transfer of a phosphate group from a donor molecule to ADP → ATP.

Q: Where does substrate-level phosphorylation occur in glycolysis?

A:

• Reaction 7: 1,3-BPG → 3-phosphoglycerate (enzyme: phosphoglycerate kinase) — first ATP yield step.

• Reaction 10: Phosphoenolpyruvate → pyruvate (enzyme: pyruvate kinase) — second ATP yield step.

Q: What other reactions occur in Stage 2?

A:

• Reaction 8: 3-phosphoglycerate → 2-phosphoglycerate (enzyme: phosphoglycerate mutase).

• Reaction 9: 2-phosphoglycerate → phosphoenolpyruvate (enzyme: enolase).

Q: How many ATP are produced in glycolysis overall?

A: 4 total ATP formed − 2 invested = net gain of 2 ATP per glucose.

Q: Why is substrate-level phosphorylation distinct from oxidative phosphorylation?

A: It does not require O₂ or ATP synthase; ATP is generated directly from high-energy intermediates.

Q: Why is the energy yield from glycolysis under anaerobic conditions considered inefficient?

A: Glycolysis produces only 2 ATP per glucose under anaerobic conditions (≈6% of the 32 ATP possible under aerobic conditions). This low yield highlights that most cellular ATP comes from oxidation of pyruvate and acetyl-CoA in mitochondria through the citrate cycle and oxidative phosphorylation.

Q: What is the overall summary of glycolysis as a metabolic pathway?

A: Glycolysis is a universal 10-step pathway that converts glucose into two molecules of pyruvate, generating a net of 2 ATP and 2 NADH. It functions under both aerobic and anaerobic conditions, links to numerous metabolic pathways, and serves as the entry point for carbohydrate catabolism and energy production.

Q: How can the regulation of a metabolic pathway be understood?

A: By examining the free energy changes (ΔG°′ and ΔG) of each reaction, which identify the steps that drive the pathway toward product formation.

Q: What does analyzing ΔG°′ and ΔG reveal about glycolysis?

A: It reveals which reactions are irreversible under physiological conditions and which are near equilibrium.

Q: Which glycolytic reactions are irreversible under normal conditions?

A: The reactions catalyzed by hexokinase, phosphofructokinase-1 (PFK-1), and pyruvate kinase — these have large negative ΔG values.

Q: What is true of the other glycolytic reactions?

A: They have small free energy changes, meaning they are near equilibrium and reversible depending on metabolite concentrations.

Q: What are the two main tasks of Stage 1 (ATP Investment) in glycolysis?

A:

1. Use ATP to generate phosphorylated intermediates that are negatively charged and trapped inside the cell.

2. Split the six-carbon compound fructose-1,6-bisphosphate (F1,6-BP) into two three-carbon sugars (G3P and DHAP), which interconvert.

Q: What are the phosphorylated intermediates in Stage 1 precursors for?

A: They are precursors to high-energy compounds 1,3-bisphosphoglycerate and phosphoenolpyruvate, used in Stage 2 for substrate-level phosphorylation.

Q: What happens in Reaction 1 of glycolysis?

A: Glucose is phosphorylated at C-6 using ATP to form glucose-6-phosphate (G6P) — activating glucose for catabolism.

Q: What type of reaction is this, energetically speaking?

A: The first ATP investment step, where the energy from ATP hydrolysis is used to transfer a phosphate group.

Q: What role does Mg²⁺ play in the reaction catalyzed by hexokinase?

A: The ATP–Mg²⁺ complex is the true substrate, shielding negative charges and stabilizing the reaction transition state.

Q: Which two enzymes catalyze glucose phosphorylation, and where are they found?

A:

• Hexokinase: present in all cells; broad substrate specificity (glucose, mannose, fructose).

• Glucokinase: present only in liver and pancreatic cells; specific for glucose.

---

Q: How do hexokinase and glucokinase differ in regulation?

A:

• Hexokinase: inhibited by product (G6P); stops when glycolytic flux is low.

• Glucokinase: not inhibited by G6P and has a lower affinity for glucose — acts as a metabolic sensor of blood glucose levels.

---

Q: How does hexokinase bind glucose?

A: Through an induced-fit mechanism that excludes water and brings ATP’s phosphoryl group close to glucose’s C-6.

Q: What structural change occurs when glucose binds to hexokinase?

A: The enzyme undergoes a large conformational change, like jaws clamping down on the substrate.

Q: What does Figure 9.21 illustrate?

A: It shows the induced-fit mechanism of hexokinase and how glucose-6-phosphate inhibits hexokinase by binding to its active site.

Q: What enzyme catalyzes the second reaction of glycolysis?

A: Phosphoglucoisomerase (also called phosphoglucose isomerase or phosphohexose isomerase).

Q: What does phosphoglucoisomerase do?

A: It interconverts an aldose (glucose-6-P) and a ketose (fructose-6-P) through opening and closing of the ring structure.

Q: What is the free energy change for this reaction, and what does it imply?

A: ΔG°′ = –2.9 kJ/mol, meaning the reaction is readily reversible and depends on intracellular concentrations of G6P and F6P.

Q: What structural change occurs in Reaction 2?

A: The six-membered ring of glucose-6-P is converted to a five-membered ring of fructose-6-P.

Q: What happens in Reaction 3 of glycolysis?

A: PFK-1 catalyzes the transfer of a phosphate from ATP to F6P, forming fructose-1,6-bisphosphate (F1,6-BP).

Q: What type of step is Reaction 3?

A: The second ATP investment reaction and a major regulatory step in glycolysis.

Q: Why is reaction 3 irreversible?

A: It has a large negative free energy change (ΔG°′ = –18.8 kJ/mol).

Q: What is the difference between bisphosphate and diphosphate compounds?

A:

• Bisphosphate: two phosphate groups on different carbons (e.g., F1,6-BP).

• Diphosphate: two phosphates covalently linked (e.g., ADP).

Q: How is phosphofructokinase-1 regulated allosterically?

• Activated by: AMP and ADP (low energy state).

• Inhibited by: ATP (high energy state).
  This ensures glycolysis runs only when energy is needed.

Q: How do the allosteric effectors of PFK-1 interact?

A: AMP, ADP, and ATP bind to the same site; AMP/ADP are positive effectors, ATP is negative.

Q: What reaction does aldolase catalyze?

A: The cleavage of fructose-1,6-BP into two 3-carbon products: glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP).

Q: What does the term “lysis” in glycolysis refer to?

A: The splitting of a 6-carbon sugar into two 3-carbon molecules — occurs in Reaction 4.

Q: What bonds are cleaved by aldolase?

A: The C-3 and C-4 bonds of fructose-1,6-BP.

Q: What does Figure 9.24 show?

A: It depicts aldolase catalyzing the cleavage of F1,6-BP between C-3 and C-4, forming G3P and DHAP. Phosphate groups on C-1 and C-6 of F1,6-BP become the phosphates of the products.

Q: What type of reaction is catalyzed by aldolase mechanistically?

A: A reverse aldol condensation, involving formation of a Schiff base intermediate with a lysine residue in the active site.

Q: What are the four steps in the aldolase mechanism?

• Fructose-1,6-BP binds to active site; lysine attacks the ketose carbon → Schiff base intermediate.

• Base abstraction by an active site carboxyl group causes C–C cleavage → first product, G3P, released.

• Isomerization forms a second Schiff base intermediate.

• Hydrolysis releases the second product, DHAP.

Q: Why is examining the free energy (ΔG°′ and ΔG) of glycolytic reactions important for understanding regulation?

A: It identifies metabolic control points—reactions with large negative ΔG values (like those catalyzed by hexokinase, PFK-1, and pyruvate kinase). These steps drive the pathway forward and are targets for regulation, while reactions with small ΔG remain near equilibrium and reversible.

Q: What is the overall summary of Stage 1 (ATP investment phase) of glycolysis?

A: Stage 1 uses two ATP molecules to phosphorylate and trap glucose, forming fructose-1,6-bisphosphate, which is then split into two 3-carbon intermediates (G3P and DHAP). This primes the pathway for Stage 2’s energy-producing reactions, setting up substrate-level phosphorylation.

Q: What does the aldolase reaction illustrate about ΔG°′ and ΔG values?

A: Under standard conditions, aldol condensation is favored because the standard free energy change is highly positive (ΔG°′ = +23.8 kJ/mol). In the cell, metabolite concentrations favor cleavage, making the actual ΔG ≈ –0.4 kJ/mol, allowing the reaction to proceed during glycolysis.

Q: Why is Reaction 5 necessary?

A: Only glyceraldehyde-3-phosphate (G3P) can continue in glycolysis, so the enzyme triose phosphate isomerase (TPI) converts dihydroxyacetone-phosphate (DHAP) into G3P.

Q: How does Reaction 5 compare to Reaction 2?

A: It’s mechanistically similar but reversed — it converts a ketose (DHAP) into an aldose (G3P) instead of the other way around.

Q: What intermediate forms during Reaction 5?

A: The reaction proceeds through formation of an enediol intermediate (Figure 9.26).

Q: What is the outcome of Reaction 5 for glycolysis as a whole?

A: It completes Stage 1 of glycolysis — after investing 2 ATP, the pathway produces two molecules of G3P per glucose phosphorylated in Reaction 1.

Q: What are the four key features of Stage 2 reactions?

A:

1. Two G3P molecules enter Stage 2 for every glucose.

2. Two substrate-level phosphorylation reactions (by phosphoglycerate kinase and pyruvate kinase) produce 4 ATP total, giving a net 2 ATP gain per glucose.

3. Two NADH molecules form via glyceraldehyde-3-P dehydrogenase, later oxidized to generate more ATP through oxidative phosphorylation (or by lactate dehydrogenase anaerobically).

4. Reaction 10 is irreversible, ensuring pyruvate is the endpoint and preventing futile cycling with gluconeogenesis.

Q: Why are separate reactions required to reverse glycolysis in gluconeogenesis?

A: The irreversible steps (e.g., Reaction 10) must be bypassed using energetically costly reactions involving ATP and GTP to synthesize phosphoenolpyruvate from pyruvate.

Q: Why is the glyceraldehyde-3-phosphate dehydrogenase reaction critical?

A: It produces 1,3-bisphosphoglycerate (1,3-BPG), a high-energy intermediate used to drive ATP synthesis.

Q: What coupled redox events occur in Reaction 6?

A: NAD⁺ is reduced to NADH while G3P is oxidized, and the released energy drives addition of inorganic phosphate (Pᵢ) to form 1,3-BPG.

Q: Why must NAD⁺ be replenished?

A: Continuous glycolytic flux requires constant NAD⁺ supply — regenerated via the electron transport chain (aerobic) or lactate dehydrogenase (anaerobic).

Q: What are the four mechanistic steps of glyceraldehyde-3-P dehydrogenase?

A:

1. G3P binds; cysteine residue forms a thiohemiacetal intermediate.

2. Aldehyde oxidized; hydride transferred to NAD⁺, producing NADH + H⁺ and forming a high-energy acyl thioester intermediate.

3. NADH leaves; replaced by new NAD⁺.

4. The acyl thioester reacts with Pᵢ to form 1,3-BPG, regenerating the enzyme.

Q: What does Figure 9.28 illustrate?

A: The detailed four-step catalytic mechanism of glyceraldehyde-3-P dehydrogenase, including hydride transfer to NAD⁺ and regeneration of the active site.

Q: What free-energy change accompanies formation of 1,3-BPG?

A: The coupled oxidation-phosphorylation yields a standard ΔG°′ of –49.4 kJ/mol, more favorable than ATP hydrolysis (ΔG°′ = –30.5 kJ/mol).

Q: How is this free-energy difference used?

A: Phosphoglycerate kinase harnesses it in Reaction 7 to phosphorylate ADP, generating ATP via substrate-level phosphorylation.

Q: Why is the hydrolysis of some phosphate compounds (like 1,3-BPG and PEP) so exergonic?

A: Because their products (e.g., 3-phosphoglycerate) have greater resonance stabilization, favoring the hydrolyzed state.

Q: How does phosphoenolpyruvate (PEP) compare energetically to 1,3-BPG?

A: PEP has an even more favorable ΔG°′ (–61.9 kJ/mol). Its hydrolysis is driven by tautomerization of pyruvate (enol ↔ keto), which stabilizes the product and makes the forward reaction highly exergonic.

Q: What do the standard free energies in Table 9.4 reveal?

• 1,3-BPG (–49.4 kJ/mol) and PEP (–61.9 kJ/mol) > ATP (–30.5 kJ/mol) in energy yield.

• Lower-energy phosphates like glucose-6-P (–13.8 kJ/mol) require ATP investment for phosphorylation in early glycolysis.

Q: What does phosphoglycerate kinase catalyze?

A: The conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate, transferring a phosphate to ADP to form ATP.

Q: Why is Reaction 7 known as the “payback reaction”?

A: It replaces the 2 ATP invested during Stage 1, restoring energy balance in glycolysis.

Q: How often does Reaction 7 occur per glucose molecule?

A: Twice — once for each G3P, producing 2 ATP total per glucose.

Q: What structural feature allows phosphoglycerate kinase to function?

A: It has two lobes, one binding ADP–Mg²⁺ and the other 1,3-BPG; substrate binding triggers a large conformational change that excludes water and aligns the phosphoryl group for transfer.

Q: How is the phosphoglycerate kinase mechanism similar to hexokinase?

A: Both use an induced-fit mechanism, bringing substrates together and excluding water to maintain a hydrophobic active site.

Q: What does Figure 9.29 show?

A: It depicts phosphoglycerate kinase catalyzing substrate-level phosphorylation, capturing the energy from 1,3-BPG to generate ATP and 3-phosphoglycerate.

Q: How do Stages 1 and 2 of glycolysis connect energetically?

A: Stage 1 invests 2 ATP to phosphorylate and split glucose into two G3P molecules, trapping energy in high-energy intermediates. Stage 2 then recovers this energy through oxidation and substrate-level phosphorylation, generating 4 ATP and 2 NADH, for a net gain of 2 ATP per glucose.

Q: How is energy flow coupled between Reactions 6 and 7 of glycolysis?

A: In Reaction 6, oxidation of G3P transfers electrons to NAD⁺ and forms 1,3-bisphosphoglycerate, storing energy in a high-energy phosphate bond. In Reaction 7, this stored energy is used by phosphoglycerate kinase to phosphorylate ADP → ATP, directly coupling oxidation to ATP synthesis.

Q: What are the actual free-energy changes for glycolytic reactions 6 and 7, and why are they significant?

A: The ΔG values are small (–1.3 kJ/mol and –3.4 kJ/mol), meaning both reactions are essentially reversible under cellular conditions.

Q: Which glycolytic reactions are irreversible, and why?

A: Reactions catalyzed by hexokinase, phosphofructokinase-1, and pyruvate kinase are irreversible due to large negative free-energy changes, making them key control points in metabolism.

Q: Why can’t small changes in metabolite concentrations reverse irreversible reactions?

A: Because the free-energy change is so unfavorable that physiological fluctuations in metabolite levels cannot shift equilibrium enough to drive reversal.

Q: How does gluconeogenesis overcome glycolysis’s irreversible steps?

A: It uses different enzymes in those steps to bypass the high-energy barriers and allow glucose synthesis from noncarbohydrate precursors.

Q: What important side reaction occurs in erythrocytes involving 1,3-bisphosphoglycerate?

A: It’s converted to 2,3-bisphosphoglycerate (2,3-BPG), an allosteric regulator of hemoglobin, linking glycolytic flux to oxygen transport.

Q: Which enzymes regulate this 2,3-BPG side pathway?

A:

• Bisphosphoglycerate mutase: converts 1,3-BPG → 2,3-BPG.

• 2,3-BPG phosphatase: converts 2,3-BPG → 3-phosphoglycerate for re-entry into glycolysis.

Q: What is the physiological result of the 2,3-BPG shunt?

A: It creates a metabolic link between glycolysis and oxygen transport by modulating hemoglobin’s affinity for O₂.

Q: Why do defects in glycolytic enzymes affect oxygen transport?

A: Because 1,3-BPG and 2,3-BPG levels depend on glycolytic activity. Defects upstream reduce 2,3-BPG, while downstream defects increase it.

Q: How do changes in 2,3-BPG levels alter hemoglobin’s oxygen affinity?

:

• ↓ 2,3-BPG → curve shifts left → higher O₂ binding (R-state stabilization).

• ↑ 2,3-BPG → curve shifts right → lower O₂ binding (T-state stabilization).

Q: What broader concept does this illustrate?

A: How biochemical enzyme defects can cause seemingly unrelated physiological conditions, such as altered oxygen delivery.

Q: What is the purpose of Reaction 8 in glycolysis?

A: To convert 3-phosphoglycerate → 2-phosphoglycerate, forming the precursor for phosphoenolpyruvate in Reaction 9 and setting up for final ATP production.