ENZYME KINETIC

M. Saifur R, PhD

Course content Enzymatic reaction Rate of Enzyme-Catalyzed Reactions Quatification of Substrate Concentration

and Reaction Rate Kinetic Parameters Are Used to Compare

Enzyme Activities Enzymes Catalyze Reactions with Two or

More Substrates Enzymes Are Subject to Reversible or

Irreversible Inhibition Enzyme Activity Depends on pH

Enzymatic Reaction

What is the catalysis.........? To answer the question, we must

understand the different between the reaction rate and equilibrium.

The catalist is affect the reaction rate but not equilbrium

E : Enzyme; S : Substrate; P : ProductEnzyme is catalist

The different of reaction rate and equilibrium

The starting point for either the forward or the reverse reaction is called the ground state. The equilibrium between S and P reflects the difference in the free energies of their ground states. The free energy of the ground state of P is lower than that of S, so G for the reaction is negative and the equilibrium favors P. The position and direction of equilibrium are not affected by any catalyst.

The rate of a reaction is dependent on an entirely different parameter.

There is an energy barrier between S and P: the energy required for alignment of reacting groups formation of transient unstable charges bond rearrangements

other transformations required for the reaction to proceed in either direction

Illustrated by the energy hill To undergo reaction, the molecules must overcome this barrier and

therefore must be raised to a higher energy level.Top of the hill transition state : a condition where the decay of S or P is equally probable.

Transition state

It is simply a fleeting molecular moment in which events such as bond breakage, bond formation, and charge development have proceeded to the precise point at which decay to either substrate or product is equally likelyThe difference between the energy levels of the ground state and the transition state is the activation energy, G‡.

Higher activation energy slower the reaction.Reaction rate can be increased by incresaing temperature or adding the catalyst.

Enzyme dcreases the activation energy

Rate of Enzyme-Catalyzed ReactionsFactor affecting the rate of a reaction catalyzed by an

enzyme : [S]

Complicated.......!!!

[S] is change because S is converted to P

Measure the initial rate (Vo), when [S] >> [E]In a typical reaction [S] = 6x [E]At the beginning of the reaction is monitored (often the first 60 seconds or less), changes in [S] can be limited to a few percent, and [S] can be regarded as constant.

Effect on Vo when [S] increases

Substrate Saturation of an Enzyme

A. Low [S] B. 50% [S] or Km C. High, saturating [S]

Leonor Michaelis and Maud Menten in 1913

The combination of an enzyme with its substrate molecule to form an ES complex is a necessary step in enzymatic catalysis.

First step E combines reversibly with S at very fast level

Second step ES complex breakdown is slow.

Rate limiting step of the recation

Quatification of Substrate Concentration and Reaction Rate

At low [S] or at the biginning of the reaction, the recation tend to [ES]At high [S], when no free E then the reaction reach to maximum or the E is saturated

What is the realtionship between the reaction rate and the [S].....?

At the biginning of the reaction : [P] is neglegible then the k-2 is ignoredIf Vo is the rate of the breakdown of ES to form a P then :

The [ES]isdifficult to measure experimentally then we need another alternative.

If Et is E total [E]t = [E]free + [ES] or [E]free = [E]t – [ES]If [S] >>Et [S] bound the E is negligible.

Rate of ES formation : k1[Et – ES][S] ..................(1)Rate of ES breakdown : k-1[ES] + k2 [ES] ...........(2)

First step:

Second step:Steady state assumption: [ES] is constant the formation of ES = breakdown of ES, then :k1[Et – ES][S] = k-1[ES] + k2[ES] ..............

(3)

Third step:

Adding the term of to both side of the Eq. 4 then

..............(4)

..............(5)

..............(6)

..............(7)

is Michaelis constant or Km

then, in term of ES, we can express Vo as :

..............(8)

..............(9)

..............(9)

Vmax is reached when the E is saturated (it means that Et = ES), so Vmax = k2[Et], then

Michaelis-Menten equation, the rate equation for a one-substrate enzyme-catalyzed reaction.

Kinetic Parameters Are Used to Compare Enzyme Activities

A double-reciprocal or Lineweaver-Burk plotMichaelis-Menten plot

Km sometimes can be used to describe the enzyme affinity to its substrate.

If k2<<k-1 then Km is reduced to k-1/k1 and is called by dissociation constant. kcat = k2 = Vmax/[Et] turn over number : the number of S molecule coverted to P at any given time on a single molecule of E whenE is saturated with S.Catalytic efficiency = kcat/Km

Enzymes Catalyze Reactions with Two or More Substrates

Enzymes Are Subject to Reversible or Irreversible Inhibition

Reversible Inhibition

Competitive : I bind to active siteUncompetitive : I bind to other site, but only to ES complexMixed (Noncompetitive) : I bind to other site, either to E or ES complex

Competitive Uncompetitive

Mixed (Noncompetitive)

Irreversible InhibitionThe irreversible inhibitors are those that bind covalently with

or destroy a functional group on an enzyme that is essential for the enzyme’s activity, or those that form a particularly stable noncovalent association.

Reaction of chymotrypsin with diisopropylfluorophosphate (DIFP) irreversibly inhibits the enzyme. This has led to the conclusion that Ser195 is the key active-site Ser residue in chymotrypsin.

Enzyme Activity Depends on pH

Enzymes have an optimum pH (or pH range) at which their activity is maximal at higher or lower pH, activity decreases.Amino acid side chains in the active site may act as weak acids and bases

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