enzymes ii kinetics · 2020. 1. 22. · 3. define velocity (initial velocity v0) and how it is...
TRANSCRIPT
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Enzymes II: kinetics الفريق الطبي األكاديمي
لكــية الطب البرشي
البلقاء التطبيقية / املركز
6102/6166أ حياها و من
Done By: - AHMAD ALSAHELE
Corrected By:-Bushra saleem
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Enzymes II:kinetics و من أحياها
6102/6166
Specific aims: Enzymes II:kinetics
1. Know what is kinetics;
2. Understand the concepts of Michaelis-Menten Kinetics and its conditions
3. Define Velocity (initial velocity V0) and how it is determined
4. Understand how the experiment of initial velocity V0 versus substrate
concentration [S ] is achieved
5. 5.Define steady state kinetics,( M-M kinetics: E and ES changes very small)
6. 6.Define Pre-steady State Kinetics and its importance:
7. Understand the concept of Vmax, and Km
8. Understand Michaelis-Menten Equation
9. Compare Michaelis-Menten kinetics enzymes with enzymes that do not
follow M-M kinetics (allosteric enzymes)
10. Describe Perfect enzymes,
11. Understand the concept of Kcat/Km
12. Know how Line weaver Burk blot is established
13. Interpret data from Line weaver Burk blot
Slide 1:
E+S >=< ES >=< ES* >=< EP >=< E+P
this equation explain that :-
substrate bind to enzyme=> to make enzyme-substrate complex=> which form
transitional state=> which give you enzyme and products=> then enzyme release
products
you should differentiate between ES & ES*
ES ==> enzyme-substrate complex (just binding)
ES* ==> the middle of the reaction (transitional state)
Michaelis-Menten kinetics
Slide 2:
E+S >=< ES >=< ES* >=< EP >=< E+P
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Enzymes II:kinetics و من أحياها
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rate of formation mean how much product produced per the unit of time on
another words the velocity of the reaction
Slide 3+4+5: Important
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Enzymes II:kinetics و من أحياها
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enzyme kinetics is very important in pharmacology and what applied to
enzyme kinetics applied to the drug kinetics
How experimenters got these data in the plot 1?
we know that by this experiment which is done in different tubes in
the first tube we add 0 substrates and in the last tube we add the
highest amount of substrate and the rest we add vary amounts of
substrate then we add a considered amount of enzyme in all tubes and
then we measure at different concentration of substrate how many
products (usually in micromole) are produced per second
when we add the enzyme to the tubes at this moment [S] decrease , [P]
increase , [E] decrease , [ES] increase these processes take place in millisecond
dramatic changes in the beginning of the reaction make measuring of velocity
of the reaction very difficult because the change in [E] & [ES] is very sharp
we can measure the velocity of the reaction when the changes is minimal this
happen after about 1 minute
measuring [P] is more important than measuring [E] or [ES]
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at the beginning of the reaction (time zero) enzyme is not added yet (the
reaction is not catalyzed by an enzyme)
at time zero [S] => high , [E] => high , [ES] => low , [P] => low
[ES] is very low at time zero because enzyme is not added to the reaction yet
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Enzymes II:kinetics و من أحياها
6102/6166
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pre-steady state:-
the state in which enzyme concentration drop rapidly
this state is not important in study of reaction velocity because [E] &
[ES] varying widely
steady state:-
we interest with this state because the study of reaction velocity in this
state is possible because [E] & [ES] relatively constant
equilibrium state:-
it is the state after the steady state in which the products will
accumulated and the reversible reactions go back and the reaction
reach equilibrium
at this period we cannot measure the velocity of reaction because the
change in [S] & [P] is very little
Slide 6:
This instrument is used to study Pre-steady State Kinetics
Can give info on reaction mechanism, rate of ES formation
Kinetic Considerations
Slide 7:
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Enzymes II:kinetics و من أحياها
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Low substrate Moderate substrate High substrate
[S] Little Good Excess
[E] Excess Enough Little
Reaction rate The reaction will be very small because the enzyme are waiting to have substrate but there is no
Reaction rate is good There is saturation so that no more product is produced per unit of time
Slide 7:
Increase in substrate concentration
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Enzymes II:kinetics و من أحياها
6102/6166
this plot explain what mentioned in the previous slide
V0 don't mean 0 time put mean steady state
when the [S] increase , V0 increase until we reach the point of saturation at
this point V0 become constant because all enzymes are busy and no more
products are produced at the unit of time
Slide 8:
the most important parameters in Michaelis-Menten kinetics are:-
V max => maximal velocity
K m => michaelis constant
line 1 represent the V max which is at very high concentration of substrate
V max depend on the amount of enzyme we have
V max is a variable for the enzymes represent enzyme availability
example: if we have two enzymes with V max 50 & 100 respectively it is not
necessary E2 stronger than E1 because [E] has an effect in V max
at point 2 all enzymes are busy and the velocity of reaction is constant
K m => the concentration of substrate at 50% of V max
K m tells the substrate affinity of the enzyme
substrate affinity (or enzyme strength) is inversely related with K m
the lowest [S] taken to reach 1/2 V max the stronger the enzyme
1
2
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Enzymes II:kinetics و من أحياها
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we called this curve hyperbolic curve and it represent all enzymes that follow
Michaelis-Menten kinetics
Slide 9:
Michaelis-Menten equation:-
Enzymes That Don’t Follow Michaelis-Menten Kinetics Include Those That Bind
Substrate Cooperatively - Binding of One Substrate Affects Binding of Others
Michaelis-Menten equation is the equation that relate between V max & K m &
[S]
we can calculate V0 at specific [S] if we know V max & K m
The unit of V max Mole/size. time
the unit of K m Mole/size
allosteric enzyme don't have hyperbolic plot instead sigmoidal
Slide 10:
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Enzymes II:kinetics و من أحياها
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allosteric enzymes:-
have a sigmoidal curve because of cooperativity
cooperativity mean if a substrate bind to one subunit this favor bind to the
second subunit
cooperativity must occur in multi subunit enzyme