7. Which of the following techniques cannot be used to quantify affinity between an enzyme and its
peptide substrate?
a. X-ray crystallography
b. Isothermal calorimetry
c. Surface plasmon resonance
d. Fluorescence anisotropy
Answer: a. X-ray crystallography
X-ray crystallography visualizes static atomic structures of enzyme-peptide substrate complexes but cannot directly quantify binding affinity (like Kd or Km) through equilibrium measurements in solution. Isothermal titration calorimetry (ITC) measures heat changes during binding to determine thermodynamic parameters including affinity constants. Surface plasmon resonance (SPR) detects real-time binding kinetics and affinity by refractive index shifts as substrate interacts with immobilized enzyme. Fluorescence anisotropy monitors rotational changes in labeled substrates upon enzyme binding, yielding quantitative affinity values from anisotropy shifts.
Option Analysis
X-ray Crystallography
This structural technique reveals enzyme-substrate binding poses at atomic resolution but provides snapshots, not dynamic affinity metrics like dissociation constants. It infers potential interactions qualitatively without equilibrium quantification.
Isothermal Titration Calorimetry (ITC)
ITC directly quantifies affinity by titrating substrate into enzyme and fitting heat data to binding isotherms, ideal for peptide substrates.
Surface Plasmon Resonance (SPR)
SPR measures association/dissociation rates for precise Kd calculation, commonly used for enzyme-peptide kinetics.
Fluorescence Anisotropy
This labels peptides with fluorophores; binding restricts rotation, increasing anisotropy proportionally to affinity for direct Kd determination.
In enzyme kinetics, the ability to quantify affinity enzyme peptide substrate interactions is crucial for understanding catalytic efficiency, measured via parameters like the Michaelis constant (Km) or dissociation constant (Kd). Lower values indicate stronger binding, vital for CSIR NET Life Sciences exam topics on biochemistry and molecular biology. This article breaks down key biophysical techniques, highlighting why X-ray crystallography falls short for quantitative affinity data while ITC, SPR, and fluorescence anisotropy excel.
Structural vs Quantitative Methods
Techniques to quantify affinity enzyme peptide substrate divide into structural (visualizing complexes) and biophysical (measuring binding strength). X-ray crystallography excels at high-resolution snapshots of enzyme-peptide poses but captures static crystal states, missing solution-phase dynamics needed for Kd or Km. Complementary methods provide direct numbers.
| Technique | Quantifies Affinity? | Key Output | Best For Peptides? |
|---|---|---|---|
| X-ray Crystallography | No | Atomic structure | Binding mode visualization |
| ITC | Yes | Kd, ΔH, ΔS | Thermodynamics in solution |
| SPR | Yes | Kd=koff/kon | Real-time kinetics |
| Fluorescence Anisotropy | Yes | Kd from anisotropy shift | Labeled small substrates |
Practical Applications in Research
ITC injects peptide substrate into enzyme, tracking heat to fit binding curves for affinity constants without labels. SPR immobilizes enzyme on a chip, flows peptide, and records sensorgrams for kinetic rates. Fluorescence anisotropy suits peptides by detecting tumbling slowdown upon binding. These enable CSIR NET-level comparisons, like competitive inhibition effects on affinity.
