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
Types of inhibition
Reversible inhibition:
- Competitive inhibition
- Non-competitive inhibition
- Uncompetitive inhibition
Irreversible (suicide) inhibition:
- Covalent modification of enzyme
Competitive inhibition
Compete for active site
Usually chemically similar to enzyme’s substrate
Competitive inhibition - the tighter the inhibitor binds…
The more substrate needed to overcome the inhibition
Competitive inhibition - when [I] = Ki…
K(app) is doubled
Competitive inhibition - what values change or remain constant
Km changes
Vmax same
k(cat) same
Overcoming competitive inhibition
Can be overcome by increasing [S]
Non-competitive inhibition
Bind to enzyme (simultaneously with substrate) at a site distant from active site or the E-S complex
Not dependent on formation of enzyme-substrate complex
Changes active site –> substrate can’t bind to active site
Non-competitive inhibition - what values change or remain constant
Km unchanged
Vmax changed - max rate decreases from [I] = Ki to [I] = 10Ki
K(cat) decreases - makes catalysis less efficient
Non-competitive inhibition - when [I] = Ki
Vmax is half what we expect
Uncompetitive inhibition
Bind to another site which is only made accessible after the substrate has bound to the enzyme
i.e. inhibitor only binds to enzyme-substrate complex
Uncompetitive inhibition - what values change or remain constant
KM change
Vmax change
Slope/gradient unchanged
Secondary plot
Ki can be calculated using a secondary plot of either:
- slopes for competitive inhibitor or
- y-axis intercepts for non-competitive inhibitor
plotted against [I]
Secondary plot - axis
x-axis: [I]
y-axis: slopes or y-axis intercepts
Inhibitor binding equation
E + I — equilibrium arrow — EI
Where forward equilibrium arrow is ka and backward equilibrium arrow is kd
Ki = [E][I] / [EI] = kd/ka
Overcoming uncompetitive inhibition
Can’t be overcome by increasing substrate concentration
Ki and inhibitor
The smaller the Ki value, the better the inhibitor
Enzyme inhibitors as drugs
Tightly binding inhibitors of key enzymes can be useful as drugs
Enzyme inhibitor as drugs - penicillin
Irreversible inhibitor of transpeptidase required for synthesis of crosslinks in peptidoglycan in bacterial cell wall
Effective mimetic of D-Ala-D-Ala peptide substrate of enzyme
Peptidoglycan - cell wall
A single, enormous, bag-shaped macromolecule because of extensive cross-linking
Transition state mimetics
Can make very good inhibitors - bind the transition state with a much higher affinity than the substrate
Unstable
Temperature dependence of a typical enzyme-catalysed reaction
During early part, rate of enzyme reaction increases exponentially
But since enzyme itself is structurally unstable at high temp, it denatures and loses catalytic activity
pH dependence of a typical enzyme-catalysed reaction
An enzyme often requires one/more of the amino acids at its active site to be charged/ionised
Thus, activity of enzyme often titrates as the charge on the amino acids change
Often an enzyme will require both a positive and negative centre at its active site to be a catalyst
This depends on pKa of ionisable amino acid side-chains, which may be shifted compared to their normal solution values
Enzymes: Quaternary structure (multimeric)
Made of more than one subunit and may have more than one active site per molecule
Allostery (co-operativity)
Occurs in multimeric enzymes
Dependence of rate on substrate conc changes
Allosteric enzymes - homotropic effect
There is communication between active sites so the binding of substrate to one active site influences further binding of substrate molecules to remaining active sites
Biological importance of allosteric enzymes
Rate of reaction is v sensitive to [S] and so can act as switches - sigmoidal dependence
Allosteric enzymes - heterotropic effect
Rate of reaction responds to presence of substances chemically unrelated to substrate
These effectors can show either a positive (activators) or negative (inhibitors) effect
Allows feedback control in metabolic pathways
Heterotropic effect - metabolic pathways
First enzyme in pathway often allosterically regulated
Last enzyme in pathway often a -ve heterotropic effect for first enzyme in pathway
Last enzyme is non-competitive since it doesn’t look chemically similar to A
Homotropic allostery - steps
Substrate binds
Conformational change around binding site
Conformational change in subunit where substrate is bound
Conformational change in other subunit
Homotropic allostery - states
T-state; high Km; low affinity for S; steeper exponential curve than normal
R-state; low Km; higher affinity for S; much less steeper exponential curve than normal
Binding of S to one subunit increases the affinity of the other subunit for S
Heterotropic allostery - steps
Inhibitor binds
Conformational change around binding site
Conformational change in subunit where inhibitor is bound
Conformational change in other subunit
Binding of I to one subunit decreases the affinity of the other subunit for S
Types of metabolic reactions
Anabolic - require energy for synthesis of complex molecules from simple precursors
Catabolic - transform fuel sources into cellular energy
General anabolic reaction equation
Energy + precursors –> complex molecules
General catabolic reaction equation
Fuel –> CO2 + H2O + energy
How does the cell transfer energy generated by catabolic process to power anabolic processes
Via ATP - acts as universal currency of free energy in biological systems
Catabolic processes make ATP and anabolic processes usually consume it
What type of reaction is ATP hydrolysis
An energy-releasing (exergonic) reaction
ATP hydrolysis equation
ATP + H2O -equilibrium arrow- ADP + Pi
Where ΔG°’ = -30.5 kJ/mol (therefore favourable and releases energy)
ATP hydrolysis - release of energy is due to…
Resonance stabilisation of free phosphate is better than tri-phosphate
Electrostatic repulsion of tri-phosphate is energetically unfavourable
Water can more effectively hydrate free phosphate than tri-phosphate
How do anabolic reactions occur
Since they tend not to be energetically spontaneous, they must be coupled to the hydrolysis of ATP, which makes the overall reaction energetically favourable i.e. ΔG°’ < 0
What does coupling reactions with ATP hydrolysis do
Shifts the equilibrium constant of the reaction
i.e. changes the equilibrium ratio of reactant and product and make the rxn more favourable
What is ‘R’
Ideal gas constant
R = 8.315 x 10^-3 kJ.mol^-1.deg^-1
What is ‘T’
Absolute temperature
T = 298K = 25°C
ΔG°’ - Standard free energy
pH = 7, i.e. [H+] = 10^-7 M
Water activity is presumed to be constant
All other reactants are at 1.0M
Pressure = 1.0 atmosphere
Redox potential (E(0)’)
A measure (in volts) of the tendency of a chemical species to acquire e- or lose e- hence be themselves reduced or oxidised
The electron-transfer potential of NADH and FADH2 is converted to…
The phosphoryl-transfer potential of ATP during oxidative phosphorylation
Can be thought of a form of free energy transfer
Faraday constant (F)
96.48 kJ.mol-1.V-1 or
96485 J.mol-1.V-1
ΔE°’
Standard redox potential at 25°C, pH = 7
Unit: V
General oxidant and reductant equation
Oxidant + e- –> reductant
Oxidants oxidise other species (and so themselves are reduced), e.g. NAD+, pyruvate, O2
Reductants reduce other species (and so themselves are oxidised), e.g. NADH, lactate, H2O
Positive and negative ΔG°’ and ΔE°’
A positive ΔE°’ will give a negative ΔG°’, which is favourable
Coupling redox reactions
Coupling a favourable redox reaction to the unfavourable reaction makes the overall reaction spontaneous, i.e. combined ΔG < 0
When [S] is much less than K(M)…
Increasing [S] won’t increase initial reaction rate (v)
What is K(M)
The max [S] required to produce Vmax / 2
Organic enzyme co-factors are often derived from…
Vitamins
Many enzymes require ______ as co-factors
Metal ions
Do allosteric enzymes obey Michaelis-Menten kinetics
No
For what types of inhibition does k(cat) change
Doesn’t change for competitive
Decreases for non-competitive and uncompetitive
Ka and Kd - relationship
Larger Ka = smaller Kd
