Lecture 8 Flashcards
Enzyme Kinetics
Why kinetics?
Some fundamental scientific reasons
• To establish the speed by which a reaction is catalyzed by an enzyme
– to understand how the enzymes in living organisms perform their functions in a timely fashion
• To see how catalysis is affected by factors like pH, ionic strength, temperature, etc. – to understand how different organisms are adapted to colder and warmer (etc.) environments
• To discover the substrate specificity of enzymes and ribozymes
• To figure out the mode of action of inhibitors – usually classified as
competitive, non-competitive and uncompetitive
• To understand accelerating or decelerating effects of non-substrate molecules on catalytic efficiency – particularly so-called allosteric effectors
• To probe in detail the catalytic mechanism of a reaction, not only by varying the substrates but also the biocatalyst itself, by e.g. protein engineering techniques and varying precisely specific side chains.
Enzyme Kinetics
Why kinetics?
Some practical reasons
• To establish the speed by which a reaction is catalyzed by an enzyme
or ribozyme – to see if a reaction is useful for industrial reactors to convert compounds into more valuable compounds
• To see how catalysis is affected by factors like pH, ionic strength, temperature, etc. – to obtain robust enzymes for bioreactors
• To discover, and modify, the substrate specificity of enzymes and ribozymes for use in bioreactors, and also as tools in biochemical and medical research
• To discover enzyme or ribozyme inhibitors in drug discovery processes, where sometimes thousands, or a few million, compounds are tested against a potential protein or RNA drug target in so-called “high-throughput screens”
• For lead optimization in drug development, i.e. optimize “leads” discovered in screens to become high affinity inhibitors with good “drug-like properties”.
Reaction rate is independent of ΔG
• ΔG describes the whether or not a reaction proceeds spontaneously and nothing about the rate of that reaction
• Some spontaneous reactions may not occur at physiological rates (e.g. peptide bond hydrolysis has a
half life of ~2500 years)
• Reaction rate depends on:
• In living systems, raising the temperature is not viable, thus, to increase reaction rates, must have ways to reduce ΔG0‡
Note: You don’t need to know this equation, but you need to know the dependence of k on ΔG0‡ and T
Enzymes increase reaction rates by lowering transition state energies
- Enzymes reduce ΔG0‡ by stabilizing the transition state of the reaction
- Reduction of transition state energy given by ΔΔG0‡ = ΔG0non - ΔG0cat
A catalyst lowers the activation energy in both directions
Enzyme Kinetic Experiment: S –> P
- Use constant amount of enzyme, [ET], where the T is for “total”.
- Measure amount of product P formed as a function of time with several initial concentrations of substrate S
- Calculate initial slopes from the graph of [P] vs. time to get initial velocities: vo = d[P]/dt
- Plot the initial velocities as function of [S]
Enzymes often form an Enzyme:Substrate complex
For many enzyme-catalyzed reactions:
• Acceleration is very fast (μsec to msec) while the time scale is in minutes.
• d[ES]/dt = 0 which means that a steady state condition applies to most of the reaction progress.
Total enzyme concentration [E]T is: [E]T = [E] + [ES]
The Michaelis-Menten Equation
The maximal velocity of a reaction, Vmax, occurs at high [S] when
the enzyme is saturated, that is, when [S]»KM
KM and Vmax are the quantities of interest
which capture the character of the catalysis of substrate S by enzyme E
Lineweaver-Burk plot
Then, this gives the Lineweaver-Burk plot with:
“ 1/[S] as the independent variable, and
“ 1/vo as the dependent variable.
This equation describes a straight line from which KM and Vmax are easy to obtain:
“ Slope: (KM/Vmax)
“ Intercept on the y-axis : 1/Vmax
“ Intercept on the 1/[S] axis: -1/KM
Competitive Inhibition
Inhibitor and Substrate compete for the Active Site
Ki is the quantity of
interest: it captures the inhibition power of inhibitor I for the particular reaction catalyzed by enzyme E.
Ki = [E][I] / [EI]
Hence: the SMALLER the Ki, the BETTER the inhibitor!
(E= enzyme, I= comp inhibitor, EI= enzyme-inhib complex)`
Uncompetitive Inhibition
Inhibitor binds to the enzyme-substrate complex, not to the free enzyme.
K’i is the quantity of interest: it captures the inhibition power of inhibitor I for the particular reaction catalyzed.
K’i = [ES][I] / [ESI]
Hence: the SMALLER the K’i, the BETTER the inhibitor!
(ES= micahaelis complex, I= uncomp inhib, ESI- enz-sub- inhib cmplex)
Uncompetitive Inhibition graph
Lineweaver-Burk Plot for Uncompetitive Inhibition: PARALLEL-LINES!!
With intersection points 1/v0= α’/Vmax on the vertical axis;
Since Vmax is known, α’ can be calculated;
And from α’, K’i can be calculated using the definition of α‘.
Mixed (noncompetitive) Inhibition
Inhibitor binds to both the enzyme-substrate complex and the free enzyme.
Protein Structure Prediction and Design
• Modern computers have allowed computational modeling of the free energy landscape of a protein
- Represent the various enthalpic and entropic effects governing folding with parameterized equations
- vdW interactions
- electrostatic interactions
- solvent entropy
- etc.
- Under Anfinsen’s hypothesis, the state of lowest free energy is the native state
- Use various optimization techniques to identify the lowest-energy state of the protein
- Design: identify a sequence that is predicted to fold into a particular state
De novo design of a novel coiled coil
• Natural coiled coils vary in their oligomerization state, but all known coiled coils are lefthanded
• Use computational design to create a right-handed coiled coils
1. Construct a backbone model for a right-handed coiled coil
2. Use sequence selection to optimize a sequence for this backbone
