Energetics and dynamics of protein action Flashcards
Hydrolase
hydrolytic cleavages
polymerase
polymerisation reactions
synthase
synthesis
kinase/phosphatases
add/remove phosphates
isomerases
rearrangements
oxide-reductases
oxidise/reduce substrates
ATPases
use 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 an endergonic reaction?
When ΔG is greater than 0
What is an exergonic reaction?
When ΔG is less than 0
What reactions tend to be spontaneous?
exergonic
How can you make an endergonic reaction spontaneous?
by coupling to with a highly exergonic 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)
When a protein binds to an enzyme why does ΔG initially fall?
because the enzyme will displace some water molecules which will increase disorder
What does the enzyme need to be complimentary to?
the transition site
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 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?
the concentration of ES stays the same (rate of formation of ES=rate of degradation of ES)
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?
velocity = 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 upper limit of enzyme efficiency and why is there an upper limit?
1x10^8M^-1.s^-1
limited by time taken for substrate to diffuse into active site
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 flipping the michaelis-menton equation over (which changes michealis-menten equation into a straight line)
What are the components of the y=mx+c equation?
y= 1/v0
m=km/Vmax
x= 1/[S]
c= 1/Vmax
What is the x intercept in a Lineweaver-Burk plot?
-1/Km
What is the y intercept?
1/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
Give one example of naturally occurring non-competitive inhibitors?
-caffeine
Give an example of a synthetic non-competitive inhibit?
haloperidol
Give an example of a naturally occurring competitive inhibitor?
digitalis
Give an example of a synthetic competitive inhibitor?
ibuprofen
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
