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
How does an increase/decrease in enzymes affect Vmax?
increase in enzyme = increase Vmax
decrease in enzyme = decrease in Vmax
How does the affinity (of enzyme and substrate) affect Km?
affinity - how specific enzymes are for substrates
increased affinity = decreased
decreased affinity = increased km
What does the y intercept and x intercept refer to on a lineweaver-burk plot?
Y intercept: 1/Vmax
X intercept: -1/km
* its negative because its on a diff (neg) quadrant
What is the slope of the lineweaver-burk plot?
Km/Vmax
What is the X and Y axis?
X: 1/[S]
Y: 1/V0
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
What are irreversible inhibitors?
- bind covalently (very strong) with or destroy a functional group on an enzyme that is essential for the enzyme’s activity
- form a particularly stable non-covalent association
- is permanent
compare diff lineweaver burt plots: competitive, uncompetitive, noncompetitive (mixed)
compare diff effect on Km: competitive, uncompetitive, noncompetitive (mixed)
competitive: increase
uncompetitive: decrease
noncompetitive (mixed): doesnt move
compare diff effect on Vmax: competitive, uncompetitive, noncompetitive (mixed)
competitive: no change
uncompetitive: decrease
noncompetitive (mixed): decrease
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 HIV and what does it cause?
Human immunodeficiency virus causes AIDS by infecting and destroying the host’s immune system
How does HIV detroy host’s immune system?
- HIV attaches to target cells and inject its genetic material
- viral RNA is transcribed to DNA using viral enzyme called reverse transcriptase
- viral proteins are made and HIV protease does further processing for viral reproduction
what can inhibitors inhibit in HIV? did it work?
reverse transcriptase and HIV protease
- inhibitors for these have been developed
- but HIV mutates rapidly and evolve drug resistance to these inhibitors
- an inhibitor “cocktail” is normally given
What is an example of an unexpected outcome in drug design?
- Pfizer scientists want to find a phosphodiesterase inhibitor to treate chest pain and high blood pressure
- studied viagra but showed no such benefits but cause side effects = erection
how can cGMP trigger relaxation?
an increase in cGMP triggers the smooth muscle cells of blood vessels to relax, and blood pressure drops
What can cGMP be hydrolysed by?
phosphodiesterase
What are the effects of phosphodiesterase?
it hydrolyses cGMP so that relaxation cannot be triggered = smooth muscle cells of blood vessels wont relax, and blood pressure wont drop
= so Pfizer scientises want to find an inhibitor for phosphodiesterase to treat chest pain and high blood pressure
How to control enzyme availability
the amount of given enzyme in a cell depends on the rate of synthesis and degredation of the enzyme
How to control enzyme activity?
an enzyme’s catalytic activity can be controlled through structural alterations that affects the enzyme’s substrate-binding affinity
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)
is the modulator that binds to an allosteric enzyme inhibitory or stimulatory?
both
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
