Enzymes as drug targets Flashcards
Enzymes
protein molecules, catalyse a specific reaction, unchanged by the reactions they catalyse
how does enzyme work as catalyst?
providing an alternative pathway for the reaction in which the rate-determining step has a lower Gibbs activation energy than that of the non-catalysed reaction
steps in enzyme kinetics;
1) reversible
2) irreversible
What is The Michaelis Menten Equations?
( π [π¬πΊ])/π π=k1 [E] [S] β(k2 - k-1 )[ES]=0
[E]0 = [ES] + [E]
π½ (ππππ ππ ππππππππ)= ( π½πππ [πΊ])/(π²π΄+[πΊ])
[S] is effectively constant
Define Vmax or (rate)max
maximum velocity the reaction can achieve. It is equal to K2[E]0
What is Km?
the Michaelis constant. It is (k2 +k-1)/k1
The velocity (rate) of an enzyme catalysed reaction increases in a __________ fashion
non-linear
When [S] is large, [S]
> > Km
Maximal Velocity Vmax
π½ (ππππ ππ ππππππππ)= ( π½πππ [πΊ])/([πΊ]) = Vmax = K2 [E]0
[s] cancel each other
What is the turnover number?
number of substrate molecules converted into product by an enzyme molecule in a unit time when the enzyme is fully saturated with substrate
what is the connection between the Vmax and k2?
Vmax and is equivalent to the rate constant k2.
turnover number equation?
K2 = Vmax/[E]0 (s-1)
turnover numbers of most enzymes with their physiological substrates fall in the range from:
1 to 10^4 s-1
KM gives an indication of how _______ the enzyme binds its substrate
tightly
Weak substrate, large KM
high [S] is needed to achieve Vmax/2 (and even more to reach Vmax!)
Good substrate, low KM:
Only a low [S] needed to reach Vmax.
Enzymes as drug targets - altered metabolic pathway
excess or deficiency of a specific metabolite.
Irreversible enzyme inhibitors: what type of bonding?
bind covalently to an enzyme,
or form a highly stable non-covalent association (tight-binding inhibitors)
name an Irreversible enzyme inhibitor?
Penicillins: irreversible enzyme inhibitors specific for a bacterial enzyme (peptidoglycan transpeptidase), needed to build the bacterial wall
name an Irreversible enzyme inhibitor?
Penicillins: irreversible enzyme inhibitors specific for a bacterial enzyme (peptidoglycan transpeptidase), needed to build the bacterial wall
Suicide inactivators or mechanism-based inactivators:
irreversible inhibitors (covalent): one drug molecule completely inactivates one enzyme molecule, and it is lost in the process
How to make inhibitors MORE selective for target? LESS side effects
1) Initially binds reversibly (Intermolecular bonding interactions), then if molecule is held long enough ο irreversible interactions take place
2) use mildly reactive electrophilic groups
Irreversible enzyme inhibitors - strength
EFFECTED by; drug concentration and time after administration we measure the enzyme activity (βhow quickly can the drug inhibit the enzyme)
Reaction of kinetics = step by step?
ADD increasing inhibitor concentrations.
- as [inhibitor] increases, the maximum amount of product formed decreases;
- as [inhibitor] increases, the inactivation rate increases
(curve of P formation drops off faster) - time passes, more enzyme molecules have been inactivated (greater deviation from uninhibited reaction)
> > > > >
good irreversible inhibitor must be specific and inactivate the enzyme rapidly
What type of bond is used for a reversible enzyme inhibitor?
non-covalent {or reversible covalent}: diffuse in and out of enzymes. Enzyme may bind its substrate (ES complex), or the inhibitor (EI complex). Once one is bound, the other may also bind (ESI complex). Only ES complex is catalytically active
KI (inhibitor dissociation constant): lower KI > _______ binding >
more effective inhibitor
tighter
Turnover Number = K2 = Vmax/[E]0 (s-1) while Km =
[S] at Β½ Vmax
list the 3 main types of reversible inhibitors?
- competitive
- uncompetitive
- mixed
Competitive inhibitors
bind the same site of the substrate (active site) and prevent the substrate from binding
Why will competitive inhibitor NOT effect Vmax?
enough substrate is present, it will out-compete the inhibitor and saturate the enzyme
Why will competitive inhibitor ^ Km?
higher [S] to saturate the enzyme when the inhibitor is present
competitive inhibitors are similar to substrates;
give example
xylose: prevents binding of glucose to hexokinase (phosphorylation of glucose hydroxymethyl group)
Xylose can make the same interactions as glucose with the active site residues, but it does not have a reactive hydroxymethyl group > it cannot react
GRAPH: As [I] increases, the curvature becomes
shallower - although all curves would reach Vmax if [S] is enough
Smaller KI > greater drop in V0
Uncompetitive inhibitors - decrease Vmax and decrease Km:
increasing [S] makes the inhibitor more effective (Vmax decreases) because it binds to ES
Uncompetitive inhibitors - Km is also decreased
apparent affinity of E for S is increased: formation of ESI complex pulls the equilibrium for ES formation to the right
uncompetitive inhibitors:
bind a different site to the substrate: anywhere on the protein, but usually not the active site
What do we call the change that happens to the active site when an uncompetitive inhibitor binds?
conformational = prevents enzyme from functioning
As uncompetitive inhibitors decrease Vmax
Km - decreases
they can easily be distinguished from competitive ones looking at the effect on enzyme kinetics:
depending on KI relative values - Vmax will always decrease, Km?
inc or decrease
kinetic behaviour present competitive and uncompetitive inhibition
mixed inhibitors
- decrease Vmax
- increase/ decrease Km
Special case of mixed inhibition: when
KI = KI
What effect does non-competitive inhibitors have on km and Vmax
- NOT effect Km
- decrease Vmax
