Energetics and Dynamics of Protein Action Flashcards
Why should we study energetics and dynamics of protein action?
Study enzyme function in health and disease
Understand complex behaviour of enzymes
Design pharmacological agents to inhibit target enzymes
Understand impact of natural and engineered gene mutations on enzyme function
What do Hydrolases do?
Hydrolytic cleavages
What do Polymerases do?
Polymerisation reactions
What do Synthases do?
Synthesis
What do Kinases and phosphatases do?
Add or remove phosphate groups
What do Isomerases do?
Rearrangements of proteins
What do Oxido-reductases do?
Oxidise / reduce substrates
What do ATPases do?
Uses ATP
What is BRENDA?
a database that tells you about proteins
What are the stages of an enzyme substrate reaction?
S+E=>ES=>EP=>E+P
What is the active site cleft?
Structural scaffold of each enzyme providing an active site.
Has catalytic activity
Has residues to interact with the substrate
What is the transition state of an enzyme?
The point with the highest amount of free energy where catalysis occurs
What is an endergonic reaction?
A reaction which requires energy and is not spontaneous since ΔG > 0
What is an exergonic reaction?
A reaction which is spontaneous and does not require energy input since ΔG < 0
How can you make an endergonic reaction spontaneous?
by coupling to with a highly exergonic reaction
How can we determine the ΔG of a reaction?
If the reaction is spontaneous or not.
How reversible the reaction is.
How does coupling an endergonic reaction with an exergonic reaction help in a metabolic pathway?
The 2 reactions can occur spontaneously
Energy is released from exergonic reaction
Used to hlp drive the endergonic reaction
What is the induced fit model?
proposes distortion of enzyme and substrate is important in catalysis
What is catalysis dependant on?
- localisation of substrate
- orientation of substrate
- binding energy of substrate
- catalytic residues on protein framework
What is the activation energy?
the energy required to reach the transition state (bent stick)
What is important about an enzymes active site?
Enzyme is complementary to the substrate when it is in its transition state
Enzyme binding brings the substrate towards its transition state
When a protein binds to an enzyme why does ΔG initially fall creating a lowered free energy of the ES*?
because the enzyme will displace some water molecules which will increase disorder (entropy) and lower the free energy
How can a structural change provide activation energy?
in hexokinase, when in open form it is unlikely to react. When glucose binds it changes its conformation to closed form which allows ATP to bind
How can conformational changes affect ΔG?
allow compensation between ΔH and ΔS to minimise ΔG
What is the michaelis menten scheme and what should be considered?
E + S <==K1==> ES ==K2==> E + P
Consider that the slowest step determines the overall rate of reaction
What is K2?
ES => E + P
Kcat
What is Kcat?
turnover number of the enzyme (how good it is) (rate that ES is converted to E + P
What is the steady state assumption?
rate of formation of ES=rate of degradation of ES so the concentration of ES stays the same.
What are the assumptions of M-M scheme?
- assume that the reverse reaction is negligible (while its favourable we can establish experimental conditions that preclude or minimise the reverse reaction)
- assume only a single central complex (ES) exists (ES breaks down directly to +P
- Assume that [S}»_space;[E] interaction of S with E does not significantly affect the concentration of S
What is Vmax?
the max velocity that can enzyme can work at
What is the equation for Vmax
Vmax=Kcat[E]
What is the Km?
the substrate conc when the reaction rate is half maximal (michaelis constant)
(measure of affinity of the enzyme for the substrate)
What is the Michaelis menten equation?
V0 = Vmax[S] / Km + [S]
How do you measure enzyme efficiency?
cat/kM
Why do you have to be careful when using Michaelis-Menton?
- does not explain kinetic properties of many enzymes
- example of sigmoidal curves for proteins with multiple subunits such as allosteric enzymes
What is the limit of enzyme efficiency and why is
there an upper limit?
Allostery
limited by time taken for substrate to diffuse into active site
What is V0?
Initial Rate of reaction
What is a problem of the michaelis menten equation?
you can’t work out Vmax because infinite amounts of substrate are needed
How do you get the Lineweaver-Burk equation?
by inversing the michaelis-menton equation over (which changes michealis-menten equation into a straight line)
How do catalysts lower the activation energy?
By stabilising the transition state
What are the components of the straight line in a line weaver burk plot and what are the axis??
y= 1/v0
m=km/Vmax
x= 1/[S]
c= 1/Vmax
Y axis = 1/Vo
X axis = 1/[S]
What is the x intercept in a Lineweaver-Burk plot?
-1/Km
What is the y intercept?
1/Vmax
What is the slope/gradient of the lineweaver-Burk plot?
Km / Vmax
What is the limitation of the Lineweaver-Burk plot?
its highly sensitive to measurements at low [S]
If it is 1/v 1/[S] graph where is high substrate conc?
At the bottom close to both axis
What happens in competitive inhibition?
- Km increases because less substrate is binding so affinity is lower
- Vmax is unaltered because if you increase [S] you can outcompete the inhibitor
How do non-competitive inhibitors work?
- molecule binds to site on enzyme other than the active site
- modifies the enzyme conformation to slow/prevent product formation
- high affinity for second binding site
What happens to the Km of non-competitive inhibition?
- Km is unaltered (substrate can still bind but can’t be converted as quickly into products Tham without the inhibitor)
- Vmax is reduced (product formation slows down
Explain why non-competitive inhibition is common in feedback inhibition, using threonine as an example.
- biosynthesis of isoleucine from threonine in bacteria involves 4 steps and 4 enzymes
- first reaction is catalysed by threonine deaminase
- this reaction is noncompetitvely inhibited by isoleucine, the end product of the pathway
- as the product increases the first reaction rate decreases
- this leads to less product and a reduction in the inhibition as the isoleucine dissociates from the enzyme
- the cycle begins again
How does the lineweaver-Burk plot show non-competitive inhibitory activity?
-1/Km stays the same (same x intercept)
1/Vmax is reduced (higher y intercept)
How does the lineweaver-Burk plot show competitive inhibitory activity?
Vmax stays the same (same Y intercept)
-1/Km increases (closer to origin)
Give a natural and synthetic non-competative inhibitor example
Natural - Caffine
Synthetic - Trichostatin
Give a natural and synthetic competative inhibitor example
Natural - Tetradotoxin
Synthetic - Ibuprofen
Explain the neuraminidase enzyme example for drug design?
Neuraminidase - essential for viral proliferation.
Sailidase - cleaves sialic acid
Sial acid held in AS cleft through a few bonds
Zanamivir (relenza) - Designed drug with a higher affinity and a competitive inhibitor binds with more bonds.
What are most drugs that inhibit enzymes?
Transition state analogues.
