Enzymes II kinetics · 2020. 1. 22. · 3. Define Velocity (initial velocity V0) and how it is...

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

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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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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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]

............................................................................

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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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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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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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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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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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

Enzymes II kinetics · 2020. 1. 22. · 3. Define Velocity (initial velocity V0) and how it is...

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