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Printable Handouts
Navigable Slide Index
- Introduction
- Why kinetics with proteins?
- Content of presentation
- Nomenclature, symbols, terms
- Thermodynamics in biochemistry
- Binding of ligands to proteins
- Thermodynamics versus kinetics
- Kinetics of protein action
- Transition state diagram
- Energy and the rate of reactions
- Energetics of enzyme catalysis
- Energy and rate constants
- A typical case
- The Michaelis-Menten mechanism
- Planning ahead
- Conventional data acquisition
- Possible loss of information
- Stopped-flow
- Stopped-flow continued
- Fast reaction
- Principle of relaxation kinetics
- Methods in relaxation kinetics
- The relaxation process
- Relaxation time and mechanisms
- Interactions without catalysis
- Rapid equlibrium assumption
- Steady-state approach
- Equilibrium vs. steady state
- Reaction progress: S and P
- Reaction progress: ES and E
- The very beginning: E and ES
- The very beginning: S and P
- The true steady state velocity
- Validity of steady state
- From data to mechanisms
- Graphical and regression analysis
- Enzyme inhibition
- The general modifier mechanism
- The specific velocity equation
- The specific velocity plot
- Primary specific velocity plot
- Secondary specific velocity plot
- Example of kinetic analysis
- Specific velocity plot - primary
- Specific velocity plot - secondary
- Inhibition profile and the final polish
- The most likely mechanism
- Not just an academic exercise
- Slow and fast-binding inhibition, slow isomerization
- Rare inhibitor species
- Progress curve for slow inhibition
- Equation fitted to data
- The delay time for inhibition
- Fast enough?
- Thanks
- References
Topics Covered
- Thermodynamics vs. kinetics
- Enzyme catalysis
- Conventional and rapid kinetic methods
- Equilibrium and steady-state approaches
- The phases of an enzymatic reaction
- Graphical and statistical analysis
- Enzyme inhibition
- The importance of kinetics for protein activity
Talk Citation
Baici, A. (2015, September 6). Kinetic analysis of protein activity [Video file]. In The Biomedical & Life Sciences Collection, Henry Stewart Talks. Retrieved December 9, 2024, from https://doi.org/10.69645/SREF5079.Export Citation (RIS)
Publication History
Financial Disclosures
- There are no commercial/financial matters to disclose.
A selection of talks on Cell Biology
Transcript
Please wait while the transcript is being prepared...
0:00
Ladies and gentlemen,
dear colleagues.
Antonio Baici from the
University of Zurich.
I guide you through an excursion
in the world of kinetics,
commented with Italian accent.
The purpose of my talk is to
discuss some topics and methods
of practical interest for the
kinetic investigation of proteins
interacting with other proteins,
ligands, and metabolites
in general, with emphasis on
the kinetics of enzyme action.
0:26
Biochemical kinetics
deals with the study
of the velocity of biologically
relevant processes.
It represents an
indispensable support
for understanding the mechanism
of action of proteins.
In fact knowing how fast
an event takes place
gives useful information
about its character.
We are aware that kinetics alone
cannot answer all questions
on the mechanism of action
of the biological process.
However, pulled information from
kinetics, structured analyses,
site-directed mutagenesis, and
other methods will do the job.
1:01
I'd like to talk about a few
concepts of practical interest.
First, I'll give you some background
on different kind of information
provided by thermodynamics
and kinetics.
Then I'd like to talk about
the kinetics of enzyme action,
starting from catalysis.
After introducing basic
methods of investigation,
I will describe two approaches
for writing kinetic equations.
Understanding the [INAUDIBLE]
of the entire progress
of an enzyme-catalyzed
reaction is a central issue
for getting a clear picture of
what is going on in kinetics.
I also try to convince you that
graphical analysis of kinetic data
is not outmoded.
With the correct use of
it, statistical analysis
using mathematical methods
explained by a powerful partner
for solving complex problems.
I conclude my talk with a
discussion of some aspects
of enzyme inhibition, focusing
on practical methods of analysis.