15 Kinetics and Inhibition Flashcards
What are the effect of pH on enzyme function?
- There is an optimal pH range for each enzyme’s function
- undergo shape changes that limit their function
e.g. shape changes on restriction enzymes limit their ability to cut restriction sites as usual
What is the effects of temperature on enzyme function?
- at high temperatures, enzymes denature and lose their shape and functionality
What are the effects of substrate conc [S] on rate of reaction?
- [S] changes during course of reaction as substrate is converted to product
- less and less as more is converted to product
- so rate will slowly plateu
- there is more product conc when there is more substrate conc
What do the dotted lines mean?
they are tangent, measures gradient, the initial velocity (rate of reaction) at time 0
How is the rate of an enzyme-catalysed reaction affected when substrate is converted to product?
the rate decreases
What is the Michaelis-Menten equation?
the rate equation for a one-substrate enzyme-catalysed reaction
* depicts the quantitative relationship between initial veolocity V0, maximum velocity Vmax, initial substrate concentration [S], Michaelis constant Km
What is Km?
Michaelis constant
* the subtrate conc [S] when veolocity is at half of its Vmax
What is the lineweaver-Burk plot?
is a plot of 1/V0 vs 1/[S] (double reciprocal of the michaelis-menten graph)
* useful in analysing enzyme inhibition, can identify what type of inhibition is happening
Why would enzyme inhibitors be one of the most important pharmaceutical agents/drugs known?
because enzymes catalyse virtually all essential cellular processes
What is the classification of diff types of inhibition?
Irreversible and reversible (competitive/uncompetitive/mixed)
What is competitive inhibition?
- competitive inhibitor competes with substrate for the active site of an enzyme
- while the inhibitor occupies the active site, it prevents binding of a substrate to the enzyme
- many are structurally similar to the substrate and combine with the enzyme without leading to catalysis
How does the Vmax and Km change with a competitive inhibitor?
Vmax = unchanged
* when [S] far exceeds [I], the probability that inhibitor will bind to enzyme is minimised and reaction exhibits normal Vmax
Km value increases in the presence of a competitive inhibitor
* Km is the [S] when 1/2 Vmax
* would need increase in [S] in order to reach 1/2 V max (half the rate of reaction)
because inhibitor blocks substrates from being formed, the rate of reaction/velocity is slower and thus takes more [S] in order to go back to the original 1/2Vmax
What is uncompetitive inhibition?
- binds at a site distinct from the substrate active site
- may not be structurally similar to the substrate
- but alters the shape of the active site
How is Vmax and Km affected by an uncompetitive inhibitor?
Vmax decreases
Km also decreases
* if Vmax decreases, the 1/2Vmax also decreases
What is mixed inhibitor?
- they bind at a site distinct from the substrate active site, but it binds to either E or ESc
How does mixed inhibitor affect Vmax and Km?
- depending on how it binds it will affect both Vmax and Km values
Explain this graph for competitive inhibition
Km increases in the presence of a competitive inhibitor
so x intercept decreases
v max remains the same so the y intercept is also the same
as the conc of inhibitor increases, the V0 decreases, since graph is inverse, it looks like its going up but the actual trend is decrease
Explain this graph for uncompetitive inhibitor
Km and Vmax are smaller, so x and y intercepts increase as the conc of inhibitor increase
graph is inverse so when x and y are increasing, the actual trend is decreasing
compare diff lineweaver burt plots: competitive, uncompetitive, noncompetitive (mixed)
The researchers working on happyase discover that the compund STRESS is potent inhibitor of happyase. Addition of 1nM STRESS increases the measured Km for SAD by a factor of 2
What type of inhibition is being observed
competitive
cuz only in competitive inhibition does Km increase
What is allosteric control?
- allosteric enzymes undergo conformational changes in response to modulator binding
- allosteric modulators bind to a different site from the active site
- the modulators may inhibit or activate the enzyme (not the same as uncompetitive and mixed inhibitors)
How are enzymes modulated by covalent modification?
- most common is protein phosphorylation and dephosphorylation
- adding and removing phosphate group of a protein
- 30% of human proteins that participate in nearly all biological processes, are subject to control by reversible phosphorylation
Is enzyme covalent modification reversible?
yes
