Enzymatic assays MCQs With Answer

Enzymatic assays MCQs With Answer

Enzymatic assays are fundamental to M.Pharm training, linking biochemical principles to practical drug development, quality control, and bioprocess monitoring. This blog provides a focused set of multiple-choice questions designed to deepen understanding of assay design, kinetics, instrumentation, and data interpretation. Questions cover Michaelis–Menten kinetics, assay formats (continuous, discontinuous, coupled), spectrophotometric and fluorometric detection, controls, units like specific activity and turnover number, and common interferences. Each MCQ is crafted to test conceptual clarity and analytical reasoning for laboratory and industrial contexts. Use these questions for revision, exam practice, or to identify topics needing further study in enzymology and bioprocess assays.

Q1. What is the primary principle behind a spectrophotometric enzymatic assay?

• Measuring heat evolved as substrate is converted to product
• Monitoring change in absorbance at a specific wavelength proportional to product or substrate concentration
• Counting enzyme molecules directly by microscopy
• Measuring pH change only without regard to absorbance

Correct Answer: Monitoring change in absorbance at a specific wavelength proportional to product or substrate concentration

Q2. In Michaelis–Menten kinetics, Km is best described as:

• The maximum velocity achieved by the enzyme
• The substrate concentration at which reaction velocity is half of Vmax
• The turnover number of the enzyme
• The inhibitor concentration that reduces velocity by half

Correct Answer: The substrate concentration at which reaction velocity is half of Vmax

Q3. Which plot linearizes Michaelis–Menten data to estimate Km and Vmax by double reciprocal?

• Semi-logarithmic plot
• Lineweaver–Burk plot
• Hanes–Woolf plot
• Eadie–Hofstee plot

Correct Answer: Lineweaver–Burk plot

Q4. What does kcat (turnover number) represent in enzymology?

• The enzyme concentration in the assay
• The number of substrate molecules converted to product per enzyme active site per unit time under saturating substrate
• The dissociation constant of enzyme-substrate complex
• The inhibitor binding rate constant

Correct Answer: The number of substrate molecules converted to product per enzyme active site per unit time under saturating substrate

Q5. Which of the following is a common chromogenic substrate for alkaline phosphatase assays?

• p-Nitrophenyl phosphate
• Bromophenol blue
• Fluorescein isothiocyanate

Correct Answer: p-Nitrophenyl phosphate

Q6. In a coupled enzymatic assay, the main purpose is to:

• Directly measure heat changes from the primary reaction
• Use a secondary reaction to produce a detectable signal from an otherwise undetectable primary reaction
• Decrease assay sensitivity to avoid saturation
• Replace cofactors in the primary reaction

Correct Answer: Use a secondary reaction to produce a detectable signal from an otherwise undetectable primary reaction

Q7. Which statement best describes a continuous enzymatic assay?

• Reaction is stopped at fixed times and product quantified later
• Product formation is followed in real time, usually by spectrophotometry or fluorometry
• Assay is performed only at a single time point
• It requires separation of product by chromatography before detection

Correct Answer: Product formation is followed in real time, usually by spectrophotometry or fluorometry

Q8. What is the International Unit (U) of enzyme activity commonly defined as?

• One mole of product formed per second
• One micromole of product formed per minute under defined conditions
• One gram of enzyme per liter
• One percent conversion of substrate per hour

Correct Answer: One micromole of product formed per minute under defined conditions

Q9. Specific activity of an enzyme preparation is expressed as:

• Units per liter (U/L)
• Units per milligram of total protein (U/mg)
• Moles of enzyme per mol substrate
• Percent purity by weight

Correct Answer: Units per milligram of total protein (U/mg)

Q10. In competitive inhibition, how are Km and Vmax affected?

• Km appears increased, Vmax unchanged
• Km decreased, Vmax decreased
• Km unchanged, Vmax decreased
• Both Km and Vmax increase

Correct Answer: Km appears increased, Vmax unchanged

Q11. Why is initial rate (v0) preferred for determining kinetic parameters?

• Because substrate depletion and product inhibition are minimal at early times
• Because it provides the largest signal at equilibrium
• Because it requires the least sensitive instruments
• Because temperature effects are negligible only at long times

Correct Answer: Because substrate depletion and product inhibition are minimal at early times

Q12. Which wavelength is commonly monitored for NADH-based dehydrogenase assays?

• 260 nm
• 280 nm
• 340 nm
• 600 nm

Correct Answer: 340 nm

Q13. The Beer–Lambert law relates absorbance to which parameters?

• Absorbance, fluorescence quantum yield, and temperature
• Absorbance, molar extinction coefficient, path length, and concentration
• Absorbance only to pH and ionic strength
• Absorbance and substrate molecular weight only

Correct Answer: Absorbance, molar extinction coefficient, path length, and concentration

Q14. Which method is most appropriate for studying rapid pre-steady-state enzyme kinetics?

• End-point colorimetric assay
• Stopped-flow spectrophotometry
• Standard spectrophotometer with manual mixing
• ELISA plate reader with 30-minute intervals

Correct Answer: Stopped-flow spectrophotometry

Q15. In an enzymatic assay validation, a blank control is used primarily to:

• Measure maximum enzyme activity
• Correct for background signal from reagents and buffer
• Calibrate the pipettes
• Increase signal-to-noise ratio by adding substrate

Correct Answer: Correct for background signal from reagents and buffer

Q16. An assay shows linear increase in absorbance only up to 2 minutes; beyond that the rate declines. The most likely cause is:

• Instrument malfunction only
• Product inhibition, substrate depletion, or enzyme instability at longer times
• Path length doubling spontaneously
• Extinction coefficient changing with time

Correct Answer: Product inhibition, substrate depletion, or enzyme instability at longer times

Q17. Which change in conditions typically increases enzyme activity until denaturation occurs?

• Increasing temperature up to an optimum
• Lowering temperature to absolute zero
• Raising pH to extreme values regardless of enzyme
• Adding strong denaturants like guanidine

Correct Answer: Increasing temperature up to an optimum

Q18. For an enzyme assay using absorbance, why is knowing the molar extinction coefficient important?

• It allows conversion of absorbance into concentration of product formed
• It defines the enzyme’s Km value
• It indicates the enzyme’s molecular weight
• It determines the assay temperature

Correct Answer: It allows conversion of absorbance into concentration of product formed

Q19. Which of the following best describes uncompetitive inhibition?

• Inhibitor binds only to free enzyme, increasing Km
• Inhibitor binds only to the enzyme–substrate complex, decreasing both Km and Vmax
• Inhibitor binds irreversibly to enzyme active site, increasing Vmax
• Inhibitor binds at an allosteric site causing only Km to decrease

Correct Answer: Inhibitor binds only to the enzyme–substrate complex, decreasing both Km and Vmax

Q20. Immobilized enzyme assays are preferred in industrial biocatalysis because they:

• Eliminate the need for cofactors entirely
• Allow enzyme reuse, improved stability, and easier separation from product
• Always increase the enzyme turnover number kcat
• Prevent any diffusion limitations regardless of particle size

Correct Answer: Allow enzyme reuse, improved stability, and easier separation from product

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