L8 - Reversible substrate binding & subsequent catalysis Flashcards
What is kinetics & enzyme kinetics?
The study of the rates of chemical reactions is called ‘kinetics’ and of enzyme catalysed reactions is called ‘enzyme kinetics’
Why study enzyme kinetics?
Can help to determine a reaction mechanism
Can provide powerful clues to an enzyme’s true biological role
Can suggest how to modify an enzyme for therapeutic purposes
Allows the rational design of novel inhibitors (eg. transition state mimics)
Stoichiometry of enzyme reactions
Overall stoichiometry: A > P
A = reactant
P = products
But may actually occur through a sequence of elementary reactions such as:
A > I1 > I2 > P
Where I1 & I2 are intermediates
What is the rate (V) of a reaction?
The instantaneous rate of appearance of P or disappearance of A is called the rate (V) of the reaction
V = d[P] / dt = - d[A]/dt
How fast the reaction is occurring
What is the rate constant (k)?
The rate of the reaction is directly related to the concentration of A by a proportionality constant, k, called the rate constant
What is a first order reaction?
Reactions where rate is directly proportional to the concentration of a single reactant are called first-order reactions
The rate constant has the units of reciprocal time (s-1)
A first order reaction is a unimolecular reaction
V = k[A]
What is a second order reaction?
Reactions that are directly proportional to the rate constant of 2 reactants are called second-order reactions
The rate constants have the units of M-1 s-1
This is a biomolecular reaction
V = k[A][B]
What are the other orders of a reaction apart from first or second?
Some reactions can be:
Pseudo-first-order (2 reactants but only 1 determines the reaction rate)
Zero-order (independent of reactant concentrations)
What is the simplest way to investigate reaction rate?
Follow the increase in reaction product over time (e.g. by spectroscopy, radiolabelling, HPLC)
When do you know equilibrium has been reached?
Eventually a time is reached when there is no net change in concentration of S or P.
E still actively converting S to P and vice versa but no overall change in concentration
What is the initial rate?
We define the rate of catalysis (V0) as the moles of product formed per unit time when the reaction is just beginning
What is Vmax?
Vmax is when enzyme is saturated and is the fastest rate at which the enzyme can work under specific conditions
Vmax is the reaction rate when the enzyme is fully saturated by substrate, indicating that all the binding sites are being constantly reoccupied
What is Michaelis-Menten kinetics?
Is one of the best-known models of enzyme kinetics
Main feature is that ES complex is a necessary intermediate in catalysis
What is K1, K-1 and K2?
They are all rate constants
K1 = the association of substrate and enzyme
K-1 = the dissociation of unaltered substrate from the enzyme
K2 = the dissociation of product from the enzyme
Assumption of steady-state
Under physiological conditions, substrate is in great excess over enzyme ([S]»_space; [E])
With the exception of the initial formation of ES*, which is usually over within milliseconds of mixing E and S, it is assumed that the system is in a steady-state
i.e. That the ES complex is being formed and broken down at the same rate, so that overall [ES] is constant until substrate is nearly exhausted:
d[ES] / dt = 0
What is Km?
The Michaelis constant
Has units of concentration & is independent of enzyme & substrate concentrations
What is the Michaelis-Menten equation?
V0 = Vmax[S] / Km + [S]
What does the Km represent?
The substrate concentration at which the reaction rate is half its maximal value
Eg. half the active sites are filled
Km = Vmax / 2
Determining KM & Vmax
Can be readily determined from rates of catalysis measured at a variety of substrate concentrations that span the KM if enzyme obeys Michaelis-Menten kinetics
Modern research uses computers to derive KM and Vmax by non-linear regression of data fit to MM model
Before computers accurate determination of KM and Vmax required algebraic manipulation of Michaelis-Menten equation to give a straight line plot
- Lineweaver-Burk Plot
What can Km tell us?
Concentration of substrate at which half the active sites are filled - thus concentration at which significant catalysis takes place
Km is an indication of the strength of the ES complex
- A high Km indicates weak binding
- A low Km indicates strong binding
What is kcat?
The catalytic constant
Number of substrate molecules converted into product by an enzyme molecule in a unit time when the enzyme is fully saturated with substrate
What is catalytic efficiency?
kcat/Km
Used as a measure of catalytic efficiency as it takes into account both rate of catalysis (kcat) and strength of enzyme-substrate interaction (KM) with a specific substrate
Can be used to compare an enzyme’s preference for several different substrates
How efficient can an enzyme be?
The upper limit for catalytic efficiency cannot exceed the maximal rate at which the enzyme can bind substrate molecules (k1)
This limit is imposed by the rate of diffusion of substrate into an enzyme’s active site
In cells enzymes are often organised into multi-enzyme complexes to get around diffusion problem – close proximity of active sites increases chance that substrate and enzyme will meet
