Related Experiment Videos
Precautions when determining kinetically the order of inactivation of enzymes by functionally irreversible inhibitors
Abstract:
The number of molecules of an irreversible inhibitor that are responsible for inactivation of a catalytic site is often deduced from the slope of a plot of the log of the apparent rate of inactivation (k') at different concentrations of inhibitor versus the log of the inhibitor concentrations. The purpose of this note is to urge caution in experimental design and interpretation if one attempts to utilize this kinetic technique to characterize the order of inactivation brought about by functionally irreversible inhibitors that initially bind reversibly to an enzyme in the process of inactivation. Representative literature cases which have utilized plots of log k' versus log [I] for this type of inactivation are discussed.
Insights
Caution is urged when using log-log plots to determine inhibitor stoichiometry for irreversible enzyme inactivation. This method may misinterpret reversible initial binding, affecting kinetic analysis of enzyme inhibitors.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Pharmacology
Background:
- Enzyme inactivation studies often use kinetic methods to determine inhibitor stoichiometry.
- Irreversible inhibitors can exhibit complex binding mechanisms, including initial reversible steps.
Purpose of the Study:
- To highlight potential pitfalls in interpreting enzyme inactivation kinetics.
- To advise caution when using log-inhibition rate versus log-inhibitor concentration plots for irreversible inhibitors with reversible initial binding.
Main Methods:
- Analysis of kinetic data from enzyme inactivation experiments.
- Review of literature employing log k' versus log [I] plots.
Main Results:
- The slope of log k' versus log [I] plots may not accurately reflect inhibitor stoichiometry when reversible binding precedes irreversible inactivation.
- Misinterpretation can arise from the initial reversible binding phase.
Conclusions:
- Experimental design and data interpretation require careful consideration for irreversible inhibitors with reversible binding steps.
- Alternative methods or careful validation are recommended for accurate stoichiometry determination in such cases.