Section 2: Enzymes Flashcards
What is an enzyme
A biological catalyst
Almost all are proteins - a few are RNA
Speed up the rate at which equilibrium is reached, but do NOT change the position of the equilibrium (i.e. make products faster, but can’t make products faster)
Types of enzymes (based on activity)
Some are fully active as just protein, but many require an associated non-protein component to show catalytic activity
For latter type, the enzyme alone is the apo enzyme, and the complete enzyme is the holo enzyme
Types of non-protein components of an enzyme
Referred to as a co-enzyme if it binds and dissociates from the protein during the catalytic cycle
Or, as a prosthetic group if it’s always bound
Enzymes: Characteristics
Very efficient catalysis
Specificity
Regulation
Enzymes: Characteristics - specificity
Generally very specific catalysts, but degree of specificity varies
Some are very specific to one reaction, whereas others may accept various chemically similar substrates
Enzymes: Characteristics - regulation
Can be controlled and regulated in various ways:
- Proteolysis of pro-enzymes - a one-way ‘on’ switch
- Proteolytic breakdown of enzymes - a one-way ‘off’ switch
- Transient covalent modification (e.g. phosphorylation) - a two-way ‘on/off’ switch
- Allosteric regulation - a graded and cooperative response to either substrate or non-substrate small molecules
What is ΔG‡
Activation required to initiate a reaction
Enzymes, ΔG and ΔG‡
Enzymes lower ΔG‡ by forming an enzyme-substrate complex, but do not change ΔG
Enzymes - amino acids
Enzymes are usually proteins containing hundreds or thousands of amino acids
Active site
Contain 3 or 4 amino acids which are catalysed in a reaction by enzymes
What are other amino acids (not the ones in active site) necessary for?
Positioning the active site amino acids in correct spatial orientation
Providing correct micro-environment for active site amino acids
Providing other sites for recognition and control purposes
k vs K
k = rate constant K = equilibrium constant
S —-> P
Substrate –> Product
S P
k(1) = forward rate constant for rxn
k(-1) = reverse rate constant for rxn
v = initial rate or velocity for rxn
K1 = ? v = ?
K(1) = [P] / [S] = k(1) / k(-1) v = k(1) [S]
Enzymes and temperatures
Enzymes are the reason living organisms can exist at moderate temperatures
In absence of efficient catalyst, some reactions would require very high temp to proceed at a measurable rate
Free energy
The energy in a physical system that can be converted to do work
Gibbs free energy (G)
The energy that can be converted into work at a uniform temp and pressure
ΔG vs ΔG‡
ΔG: The overall free energy change in a rxn
ΔG‡: The Ea required to initiate a rxn
Binding energy
The free energy that is released by the formation of weak bonds between substrate and enzyme
Maximised when substrate is in transition state
What does the transition state represent
The tightest interaction between substrate and enzyme
However, it is the least stable chemical form of the substrate
Can be thought of as the moment where the bond decides if it will break or reform
Enzymes have evolved to…
Recognise the transition state of the chemical rxn they catalyse
Promiscuity (moonlighting)
May be key to redundancy, resilience and adaptability in biological systems
Lock and key theories
Induced fit - there is some flexibility in enzymes
Conformation selection - will have a range of substrates, and the right one will bind
What does enzyme kinetic analysis tell us
How fast enzymes will go
How much substrate is needed to go at a particular speed
Also key to enzyme inhibition
Why is enzyme inhibition important
Important to understand metabolic regulation and action of drugs
Basic kinetic enzyme model
Established by Leonar Michaelis and Maud Menten, who proposed the simplest possible reaction scheme is this:
E + S -equilibrium arrow- ES —k(cat)—> E + P
Where forward equilibrium arrow is k(a) and backward equilibrium arrow is k(d)
2 step process
k(a), k(d) and k(cat)
k(a) = association k(d) = dissociation k(cat) = rate limiting step
Michaelis-Menten model - assumptions
Catalyst is the slowest step
Much more substrate than enzyme
Conc of enzyme-substrate complex is constant
Reverse reaction is negligible
As long as these assumptions are met, the equation will predict the behaviour of the enzyme
Protein and ligand - equation
Protein + Ligand -eq arrow- Protein-ligand
Forward equilibrium arrow is Ka
Backward equilibrium arrow is Kd
Protein and ligand - K(d) = ?
[P][L] / [P-L] = Kd / Ka
Units: M
When K(d) = [L], half is in complex, half is free
K(M) = ?
{ Kd + Kcat } / Ka
When [S] = Km, reaction velocity is half of Vmax
What is the Michaelis-Menten equation for
Allows us to predict the rate of an enzyme reaction at any [S] if we know the Vmax and K(M)
Accurate values of Vmax and Km can be best calculated by least-squares fitting of the Michaelis-Menten equation
Lineweaver-Burk double-reciprocal plot - axis
x-axis: 1/[S]
y-axis: 1/v
Lineweaver-Burk double-reciprocal plot - points
Where line hits x-axis = -1/Km
Where line hits y-axis = 1/Vmax
Gradient of line = Km/Vmax
Protein and ligand - [Ligand] = ?
[Ligand] = Kd when half of protein is occupied by ligand θ
Kd and affinity
A smaller Kd means a higher affinity
Michaelis-Menten model - the enzyme catalyses…
The conversion of substrate to product
Michaelis-Menten model - The steady state assumption
The rate of formation of the enzyme-substrate complex is equal to the rate of its breakdown
Therefore [ES] remains constant even if [S] changes
