Substrate phosphorylation MCQs With Answer

Substrate phosphorylation MCQs With Answer is an essential study tool for B. Pharm students preparing for pharmacology, biochemistry, and metabolism exams. This concise, keyword-rich introduction covers substrate-level phosphorylation vs oxidative phosphorylation, key enzymes (phosphoglycerate kinase, pyruvate kinase, succinyl-CoA synthetase), cellular locations (cytosol and mitochondrial matrix), energetics, regulation, and clinical relevance such as pyruvate kinase deficiency and anaerobic ATP production. These substrate phosphorylation MCQs help strengthen concepts in glycolysis, TCA cycle, and fermentation while improving problem-solving for ATP generation questions. Use these focused substrate-level phosphorylation questions and answers to revise mechanisms, regulators, and experimental evidence effectively. Now let’s test your knowledge with 50 MCQs on this topic.

Q1. Which reaction is a classic example of substrate-level phosphorylation?

• PEP to pyruvate by pyruvate kinase producing ATP
• Oxidation of NADH at Complex I
• ATP synthesis by ATP synthase driven by proton gradient
• Electron transfer from cytochrome c to oxygen

Correct Answer: PEP to pyruvate by pyruvate kinase producing ATP

Q2. Which glycolytic intermediate donates a phosphate for substrate-level phosphorylation catalyzed by phosphoglycerate kinase?

• 3-phosphoglycerate
• 1,3-bisphosphoglycerate
• Fructose 1,6-bisphosphate
• Glucose-6-phosphate

Correct Answer: 1,3-bisphosphoglycerate

Q3. In the citric acid cycle, substrate-level phosphorylation occurs during which conversion?

• Isocitrate to α-ketoglutarate
• Succinyl-CoA to succinate producing GTP
• Succinate to fumarate
• Malate to oxaloacetate

Correct Answer: Succinyl-CoA to succinate producing GTP

Q4. Where does substrate-level phosphorylation occur in eukaryotic cells?

• Inner mitochondrial membrane exclusively
• Cytosol and mitochondrial matrix
• Intermembrane space only
• Lysosomal lumen

Correct Answer: Cytosol and mitochondrial matrix

Q5. Which statement best distinguishes substrate-level phosphorylation from oxidative phosphorylation?

• Substrate-level uses proton gradient; oxidative uses direct phosphate transfer
• Substrate-level is direct phosphate transfer to ADP; oxidative depends on chemiosmotic proton gradient
• Both require ATP synthase to produce ATP
• Only oxidative phosphorylation occurs in the cytosol

Correct Answer: Substrate-level is direct phosphate transfer to ADP; oxidative depends on chemiosmotic proton gradient

Q6. During anaerobic glycolysis in muscle, which process supplies most of the ATP?

• Oxidative phosphorylation in mitochondria
• Substrate-level phosphorylation in glycolysis
• ATP synthesis by mitochondrial ATP synthase driven by oxygen
• Electron transport chain complex IV activity

Correct Answer: Substrate-level phosphorylation in glycolysis

Q7. Which enzyme deficiency is classically associated with reduced substrate-level phosphorylation in red blood cells leading to hemolytic anemia?

• Hexokinase deficiency
• Phosphofructokinase deficiency
• Pyruvate kinase deficiency
• Glucose-6-phosphate dehydrogenase deficiency

Correct Answer: Pyruvate kinase deficiency

Q8. How many ATP molecules are produced by substrate-level phosphorylation during glycolysis per molecule of glucose (gross yield)?

• 1 ATP
• 2 ATP
• 4 ATP
• 36 ATP

Correct Answer: 4 ATP

Q9. Which high-energy intermediate has a greater phosphoryl-transfer potential than ATP and donates phosphate in substrate-level phosphorylation?

• Glucose-6-phosphate
• Phosphoenolpyruvate (PEP)
• Pyruvate
• Citrate

Correct Answer: Phosphoenolpyruvate (PEP)

Q10. Which pair of glycolytic enzymes catalyze substrate-level phosphorylation?

• Hexokinase and phosphofructokinase
• Phosphoglycerate kinase and pyruvate kinase
• Glyceraldehyde-3-phosphate dehydrogenase and enolase
• Aldolase and triose phosphate isomerase

Correct Answer: Phosphoglycerate kinase and pyruvate kinase

Q11. Which cofactor is directly required to form the high-energy intermediate 1,3-bisphosphoglycerate that enables subsequent substrate-level phosphorylation?

• FAD
• NAD+
• Coenzyme A
• Biotin

Correct Answer: NAD+

Q12. Which of the following best describes the energy source for substrate-level phosphorylation?

• Energy from the proton motive force across inner mitochondrial membrane
• Energy released from hydrolysis of a high-energy phosphate group of a metabolic intermediate
• Energy from light absorbed by chlorophyll
• Energy from sodium ion gradient across plasma membrane

Correct Answer: Energy released from hydrolysis of a high-energy phosphate group of a metabolic intermediate

Q13. What effect does arsenate have on ATP yield from glycolysis?

• Increases ATP yield by stabilizing intermediates
• Has no effect on ATP yield
• Decreases ATP yield by bypassing 1,3-BPG formation
• Enhances oxidative phosphorylation

Correct Answer: Decreases ATP yield by bypassing 1,3-BPG formation

Q14. Which pathway relies exclusively on substrate-level phosphorylation for ATP production?

• Oxidative phosphorylation
• Fermentation
• Electron transport chain-driven phosphorylation
• Photosynthetic photophosphorylation

Correct Answer: Fermentation

Q15. Which molecule acts as the immediate phosphate acceptor in substrate-level phosphorylation reactions?

• AMP
• ADP or GDP
• ATP
• cAMP

Correct Answer: ADP or GDP

Q16. Which experimental observation would indicate ATP production is from substrate-level phosphorylation rather than oxidative phosphorylation?

• ATP production stops when respiration is inhibited by cyanide
• ATP production persists after inhibition of the electron transport chain
• ATP synthesis increases with addition of an uncoupler that collapses proton gradient
• ATP production requires intact inner mitochondrial membrane potential

Correct Answer: ATP production persists after inhibition of the electron transport chain

Q17. Which of the following is NOT a substrate-level phosphorylation reaction?

• Conversion of 1,3-BPG to 3-PG with ATP formation
• Conversion of PEP to pyruvate with ATP formation
• ATP formation by ATP synthase using proton gradient
• Succinyl-CoA to succinate producing GTP

Correct Answer: ATP formation by ATP synthase using proton gradient

Q18. Which regulator activates pyruvate kinase in glycolysis, promoting substrate-level phosphorylation?

• ATP
• Alanine
• Fructose 1,6-bisphosphate
• Citrate

Correct Answer: Fructose 1,6-bisphosphate

Q19. How many substrate-level phosphorylation steps are there in glycolysis per glyceraldehyde-3-phosphate molecule?

• 0
• 1
• 2
• 3

Correct Answer: 2

Q20. Which metabolic condition increases the reliance on substrate-level phosphorylation for ATP?

• Hyperoxia
• Hypoxia or impaired mitochondrial function
• Aerobic rest state
• Photosynthesis-active state

Correct Answer: Hypoxia or impaired mitochondrial function

Q21. In the reaction catalyzed by succinyl-CoA synthetase, which nucleoside triphosphate is typically produced in many tissues?

• ATP exclusively
• GTP (guanosine triphosphate)
• CTP
• UTP

Correct Answer: GTP (guanosine triphosphate)

Q22. Which statement about substrate-level phosphorylation is TRUE?

• It always requires oxygen
• It directly transfers a phosphoryl group from substrate to ADP or GDP
• It occurs only in mitochondria
• It uses ATP synthase to form ATP

Correct Answer: It directly transfers a phosphoryl group from substrate to ADP or GDP

Q23. Which glycolytic enzyme generates the high-energy intermediate that precedes the second substrate-level phosphorylation?

• Enolase producing PEP
• Triose phosphate isomerase producing DHAP
• Hexokinase producing G6P
• Aldolase producing FBP

Correct Answer: Enolase producing PEP

Q24. Which of these best explains why PEP can phosphorylate ADP to ATP?

• PEP has a lower free energy of hydrolysis than ATP
• PEP binds to ATP synthase directly
• PEP hydrolysis releases more free energy than ATP hydrolysis, driving phosphate transfer
• PEP is converted to oxaloacetate first

Correct Answer: PEP hydrolysis releases more free energy than ATP hydrolysis, driving phosphate transfer

Q25. Which inhibitor would reduce ATP produced by substrate-level phosphorylation in the TCA cycle?

• Fluoroacetate inhibiting aconitase step
• Cyanide inhibiting complex IV of ETC
• Methylene blue inhibiting NADH dehydrogenase
• Arsenate bypassing 1,3-BPG formation in glycolysis

Correct Answer: Fluoroacetate inhibiting aconitase step

Q26. In which cellular compartment is succinyl-CoA synthetase located where its substrate-level phosphorylation occurs?

• Cytosol
• Mitochondrial matrix
• Inner mitochondrial membrane intermembrane space
• Golgi lumen

Correct Answer: Mitochondrial matrix

Q27. Which of the following best describes the role of substrate-level phosphorylation during early intense exercise?

• It buffers ATP supply rapidly when oxidative phosphorylation is not yet ramped up
• It is the primary source of ATP after 30 minutes of exercise
• It depends on oxygen to increase ATP production
• It synthesizes ATP via ATP synthase

Correct Answer: It buffers ATP supply rapidly when oxidative phosphorylation is not yet ramped up

Q28. Which metabolic intermediate formed by glyceraldehyde-3-phosphate dehydrogenase sets up substrate-level phosphorylation?

• 3-phosphoglycerate
• 1,3-bisphosphoglycerate
• 2-phosphoglycerate
• Fructose-6-phosphate

Correct Answer: 1,3-bisphosphoglycerate

Q29. If a researcher adds cyanide to cultured cells, which ATP source remains initially available?

• Oxidative phosphorylation
• Substrate-level phosphorylation in glycolysis
• ATP synthase-driven phosphorylation
• Mitochondrial electron transport chain ATP production

Correct Answer: Substrate-level phosphorylation in glycolysis

Q30. Which of the following best characterizes a “high phosphoryl-transfer potential” compound?

• Compound that hydrolyzes with less negative ΔG than ATP hydrolysis
• Compound that cannot donate phosphate to ADP
• Compound whose phosphate hydrolysis releases more free energy than ATP hydrolysis
• Compound located only in peroxisomes

Correct Answer: Compound whose phosphate hydrolysis releases more free energy than ATP hydrolysis

Q31. Which process will NOT be directly affected by inhibition of phosphoglycerate kinase?

• ATP formation in glycolysis at the 1,3-BPG step
• Conversion of 1,3-BPG to 3-PG
• Electron transport chain proton pumping
• Substrate-level phosphorylation in glycolysis

Correct Answer: Electron transport chain proton pumping

Q32. Which statement about substrate-level phosphorylation in bacteria is correct?

• Bacteria exclusively use oxidative phosphorylation for ATP
• Many bacteria generate ATP via substrate-level phosphorylation during fermentation
• Substrate-level phosphorylation in bacteria requires mitochondria
• Bacterial substrate-level phosphorylation depends on chloroplasts

Correct Answer: Many bacteria generate ATP via substrate-level phosphorylation during fermentation

Q33. Which enzymatic activity directly forms ATP from ADP by transferring a phosphate group from phosphoenolpyruvate?

• Pyruvate dehydrogenase
• Pyruvate kinase
• Enolase
• Phosphofructokinase

Correct Answer: Pyruvate kinase

Q34. In eukaryotic cells with defective electron transport chain, which metabolite accumulates leading to increased substrate-level phosphorylation via glycolysis?

• ATP
• NADH, causing increased lactate production and glycolytic flux
• Oxygen
• Citrate exclusively

Correct Answer: NADH, causing increased lactate production and glycolytic flux

Q35. Which of the following is a regulatory effect that decreases substrate-level phosphorylation in glycolysis?

• High levels of fructose 1,6-bisphosphate
• High ATP concentrations inhibiting phosphofructokinase and pyruvate kinase
• High ADP levels activating pyruvate kinase
• Increase in AMP activating glycolysis

Correct Answer: High ATP concentrations inhibiting phosphofructokinase and pyruvate kinase

Q36. Which of the following accurately reflects ATP yield differences?

• Substrate-level phosphorylation yields more ATP per glucose than oxidative phosphorylation
• Oxidative phosphorylation yields the majority of ATP per glucose compared to substrate-level phosphorylation
• Both processes yield equal ATP per glucose under aerobic conditions
• Substrate-level phosphorylation yields no ATP

Correct Answer: Oxidative phosphorylation yields the majority of ATP per glucose compared to substrate-level phosphorylation

Q37. Which metabolic enzyme produces ATP (or GTP) directly by substrate-level phosphorylation in the TCA cycle?

• Isocitrate dehydrogenase
• α-Ketoglutarate dehydrogenase
• Succinyl-CoA synthetase
• Malate dehydrogenase

Correct Answer: Succinyl-CoA synthetase

Q38. Which of the following is a direct experiment to demonstrate substrate-level phosphorylation in isolated mitochondria?

• Measure ATP production after adding succinate when respiratory chain is inhibited
• Measure oxygen consumption only
• Measure ATP after adding oligomycin (ATP synthase inhibitor) and the metabolic substrate for succinyl-CoA synthetase
• Observe proton gradient with no ATP measurement

Correct Answer: Measure ATP after adding oligomycin (ATP synthase inhibitor) and the metabolic substrate for succinyl-CoA synthetase

Q39. Which molecule’s hydrolysis releases more free energy than ATP hydrolysis, enabling substrate-level phosphorylation in glycolysis?

• Glucose-6-phosphate
• Phosphoenolpyruvate (PEP)
• Ribose-5-phosphate
• Pyruvate carboxylate

Correct Answer: Phosphoenolpyruvate (PEP)

Q40. Which of these is a major physiological role of substrate-level phosphorylation?

• Generate majority of ATP during aerobic rest
• Provide rapid ATP supply under anaerobic or hypoxic conditions
• Transfer electrons to oxygen
• Generate proton motive force

Correct Answer: Provide rapid ATP supply under anaerobic or hypoxic conditions

Q41. Which step in glycolysis consumes ATP rather than producing it?

• 1,3-BPG to 3-PG
• PEP to pyruvate
• Hexokinase conversion of glucose to glucose-6-phosphate
• Succinyl-CoA to succinate

Correct Answer: Hexokinase conversion of glucose to glucose-6-phosphate

Q42. Which of the following best explains why substrate-level phosphorylation is important in anaerobic organisms?

• They have highly efficient electron transport chains
• They cannot use oxidative phosphorylation due to lack of terminal electron acceptor and rely on substrate-level phosphorylation for ATP
• They produce proton gradients that drive ATP synthase more strongly
• They synthesize ATP via photosystem II

Correct Answer: They cannot use oxidative phosphorylation due to lack of terminal electron acceptor and rely on substrate-level phosphorylation for ATP

Q43. In erythrocytes lacking mitochondria, ATP is produced mainly by which mechanism?

• Oxidative phosphorylation
• Substrate-level phosphorylation in glycolysis
• Creatine kinase in mitochondria
• Photosynthetic photophosphorylation

Correct Answer: Substrate-level phosphorylation in glycolysis

Q44. Which metabolic inhibitor specifically prevents formation of ATP by ATP synthase but leaves substrate-level phosphorylation intact?

• Oligomycin
• Fluoride
• Arsenate
• Rotenone

Correct Answer: Oligomycin

Q45. Which clinical scenario would increase glycolytic substrate-level phosphorylation in affected tissues?

• Hyperventilation with excess oxygen
• Ischemia causing reduced oxygen delivery
• Enhanced mitochondrial biogenesis
• Administration of uncouplers increasing ATP yield

Correct Answer: Ischemia causing reduced oxygen delivery

Q46. Which of the following is TRUE regarding the energetics of substrate-level phosphorylation?

• It always yields more ATP per step than oxidative phosphorylation
• It involves direct transfer of a phosphate group from a substrate with sufficient free energy of hydrolysis to ADP
• It requires ATP synthase and a proton gradient
• It cannot operate in the presence of oxygen

Correct Answer: It involves direct transfer of a phosphate group from a substrate with sufficient free energy of hydrolysis to ADP

Q47. Which product increase indicates enhanced substrate-level phosphorylation during anaerobic glycolysis?

• Decrease in lactate formation
• Increase in lactate production and net ATP from glycolysis
• Increase in oxidative phosphorylation rate
• Decrease in NAD+ regeneration

Correct Answer: Increase in lactate production and net ATP from glycolysis

Q48. Which enzyme catalyzes the conversion that directly yields ATP by transferring phosphate from 1,3-BPG?

• Phosphoglycerate kinase (PGK)
• Glyceraldehyde-3-phosphate dehydrogenase
• Phosphoglycerate mutase
• Enolase

Correct Answer: Phosphoglycerate kinase (PGK)

Q49. Which of the following observations would reduce net ATP yield from glycolysis by removing substrate-level phosphorylation?

• Inhibition of hexokinase
• Inhibition of phosphoglycerate kinase or pyruvate kinase
• Activation of pyruvate kinase
• Increased NAD+ regeneration

Correct Answer: Inhibition of phosphoglycerate kinase or pyruvate kinase

Q50. Which experimental result supports the chemiosmotic mechanism for oxidative phosphorylation, distinguishing it from substrate-level phosphorylation?

• ATP synthesis continues when proton gradient is collapsed by an uncoupler
• ATP synthesis by ATP synthase stops when proton gradient is collapsed by an uncoupler while substrate-level phosphorylation persists
• Substrate-level phosphorylation requires proton gradient collapse to proceed
• Electron transport proceeds only in the cytosol

Correct Answer: ATP synthesis by ATP synthase stops when proton gradient is collapsed by an uncoupler while substrate-level phosphorylation persists

G S Sachin

I am a Registered Pharmacist under the Pharmacy Act, 1948, and the founder of PharmacyFreak.com. I hold a Bachelor of Pharmacy degree from Rungta College of Pharmaceutical Science and Research. With a strong academic foundation and practical knowledge, I am committed to providing accurate, easy-to-understand content to support pharmacy students and professionals. My aim is to make complex pharmaceutical concepts accessible and useful for real-world application.

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