Allostery Flashcards
Allostery
Binding of one ligand to enzyme/protein is affected by the binding of another at a different site
Enzyme States
T state: inhibitor bound
R state: add activator/substrate bound
Feedback Inhibition
End product of metabolic pathway inhibits starting enzymes in pathway to prevent excess production
- negative regulation
Aspartate Transcarbamylase
Catalyses committed step in pyrimidine biosynthesis
3 regulatory dimers (heterotropic) and 2 catalytic trimers (homotropic)
Controlled by feedback inhibition
- inhibited by CTP which is the final product of the pathway to ensure pyrimidines are not needlessly synthesized
Active site contains residues from more than one subunit to form high affinity substrate site - change to R state shifts Lysine and serine residues into place to form ionic interactions with the substrate
Regulating of ATCase
CTP (cytidine triphosphate) is structurally different to substrate so binds to an allosteric site in regulatory subunit
CTP binding stabilises the T form, decreasing enzyme activity
Increase L (allosteric coefficient)
Binding makes it more difficult fir substrate binding to convert enzyme to R state, ie. more substrate is needed to obtain a specific rate
Sigmoid curve is shifted right
Kinetics of ATCase allostery
Sigmoid curve caused by cooperativity
Catalytic subunits follow MM kinetics
PALA
Competitive inhibitor of ATCase
- similar to the reaction intermediate stabilised by the enzyme but more chemically stable so that the ES complex is able to be studied
Cooperativity
Binding of ligand at one site affects the binding at other sites
Sigmoidal curve: means enzyme activity is more sensitive to changes in substrate concentration near Km, creating a ‘threshold’ effect
R and T state Kinetics
Exist in equilibrium: T state is energetically more stable so in the absence of substrate predominates
T: low substrate affinity and low catalysis
R: high substrate affinity and high catalysis
Presence of additional substrate increases the fraction of enzyme needed in the more active R state- the eq. position depends on the number of occupied active sites
Allosteric Coefficient
T/R ratio
Homotropic Effects
effects of substrates on allosteric enzymes
- molecule causing the cooperativity is the one affected by it
Concerted Model
Change in enzyme from T to R state is all or none
Sequential Model
Ligand binding affects other sites without causing all subunits to undergo T to R transition
Heterotropic Effects
effect of non-substrate molecules on allosteric enzymes
Symmetrical (concerted) model of MWC
- oligomeric
- T dominant when ligand absent but has low affinity for ligand
- molecular symmetry in oligomer is conserved: all or none effect
- free energy of binding stabilises R relative to T state
Sequential KNF model
- induced fit of binding site
- subunits can be in different states
- conformational change not fully propogated
- intermediat of half R/half T
- one conformational state in ligand absence
- one conformational state in ligand absence
- conformational change sequential on ligand binding
- interactions between subunits negative and positive
- different dissociation constants
MWC Parameters
- number of binding sites
- ratio of concentrations of T/R states in ligand absence
- affinity of sites in R state for ligand binding
- measure of how much more tightly subunits in R state bind compared to T state
c parameter
dissociation constant of R / dissociation constant of T
-smaller value = more dramatic change
Hill Equilibrium
Assumes a single Keq to describe allosteric behaviour
Ratio of concentrations of T and R states is reduced by a factor of c for every ligand that binds
Hill Plot
Slope gives Hill coefficient measuring cooperativity of binding
K vs. V systems
K:
- almost all enzymes are K systems
- binding affinity is affected; catalytic subunits rotate to form ionic interactions with substrate to stabilise binding
- Vmax constant
V:
- allosteric control modifying chemical rate but not affinity
- affecting rate after ES complex is formed
Negative Cooperativity
Binding affinity declines
Modulating Paramaters of MWC Model:
L: larger value means T/R equilibrium lies to T state (curve shift right)
c: smaller value means greater affinity of substrate for R state/greater difference in binding to the T and R forms
n: higher value means more substrate can bind and therefore T/R equilibrium is more shifted to R state (more cooperative)
Myoglobin vs Hemoglobin
Myoglobin: high affinity curve (hyperbolic)
Hemoglobin: sigmoid curve (weaker affinity)
- divergent evolution
Iron Ligation in Heme
- heme is in planar porphyrin ring coordinated by 4 nitrogen atoms
- proximal histidine is one ligand
- oxygen is final ligand
Heme Doming
Without oxygen binding, the VDW radius of the iron atom is too large so it is below the plane of the ring: this is heme transport
Oxygen Binding
- oxygen binding causes VDW radius to shrink because of d orbital collapse
- this change in planarity is the driving force of structural change in the protein
Bound O2 resonance
Fe2+ ferrous ion
Fe3+ ferric ion doesn’t bind oxygen creates superoxide toxic ion
Cooperative Binding of oxygen to Hemoglobin
- sigmoid curve
- allows effective release of oxygen when it is needed
- plot fractional saturation against partial pressure of oxygen
- increased pressure of oxygen causes a shift to R state, creating a sigmoidal curve
Why use hemoglobin
- increases O2 carrying capacity compared to water
- increased binding sites and molecular stabilisation
Quaternary Structure of Hb
- a pair of aB dimers
- a1B1 and a2B2
Hill Equilibrium
X + nL –> X(L)n
- only 1 parameter
- describes kinetics
- assumes a chemical equilibrium is present the binds more than one ligand all at the same time
- doesn’t describe extends of sigmoidal curve
- examine the number of bound ligands at the same time
Allostery Summary
- almost always in T state with weak binding sites
- at low substrate concentration the protein-ligand complex dissociate rapidly
- increased substrate concentration and the weak binding site starts to fall on average
- in MWC model, the molecule favors R state formation
- molecule switches to R state and additional binding sites become high affinity
- substrate concentration high relative to this affinity - all sites immediately filled
Hill Coefficient
- ‘n’
- measure of cooperativity
- linear rise on graph
- when n = number of binding sites, the cooperativity is maximal
- determined by the affinity difference between states
Hill Plots for Myoglobin and Hemoglobin
nH: measure of cooperativity <1 : negative cooperativity =1: no cooperativity >1: positive cooperativity - myoglobin has no cooperativity - hemoglobin has positive cooperativity except at very high or very low substrate concentrations in which the molecule exists solely in the T or R states
Allosteric Model of Oxygen Binding to Hb
- requires both symmetry and sequential models
- minimum 2 subunits need to bind oxygen for a T-R transition
- structural transition of iron is transmitted directly to other subunits
Structural T-R transition in Hb
- iron atom moves into the place of the heme when oxygen binds
- helix f contains histidine ligand moves
- movement of helix alter the interface between aB pairs
- aB pairs slide and rotate upon formation of the R state
ie. part of the free energy of oxygen binding in the active site is transmitted into force driving T-R transition
- enough energy to break salt bridges holding enzyme int the T form
BPG Regulation
- heterotropic allosteric inhibitor
- product of a shunt pathway in glycolysis
- promotes oxygen release from Hb
- occupies central hole in T form of Hb, so stabilizes T form until oxygen concentration become higher
- decreases affinity of Hb for oxygen
- dissociation curve shifted to lower pO2
- higher altitudes rapidly increase BPG levels, decreasing O2 binding affinity, increasing O2 unloading in the capillaries
Fetal Hb
- reduced affinity for BPG
- higher affinity for oxygen than maternal Hb
allows effective transfer to them
Bohr Effect
- increasing carbon dioxide levels promotes oxygen release
- protonation of histidine due to lowering pH promotes stabilisation of T form of deoxyhemoglobin
- CO2 also forms salt bridges with amino acid backbones to stabilise the T state
Result: respiring tissue = CO2 production = weak acid decreases pH = stabilises T state = oxygen release to these tissues
Sickle Cell Anemia
- genetic mutation in B subunit promoting aggregation into insoluble fibers
- essential locked into T state
- can sometimes provide an evolutionary advantage for malaria infection
