Enzymes

• The secret ingredient in living organism is Catalysis, a process performed by protein enzymes. • Their three-dimensional architecture gives them exquisite specificity to select the substrate molecules to which they will bind and on which they will operate. • The scene of operation called “active site” is usually a groove ,cleft or cavity on the surface of the protein. • Enzyme function frequently occurs many times, and in some cases many thousands of times per second.

• The miracle of life: a myriad chemical reactions in the cell occur simultaneously with great accuracy and at astonishing speed. • Without the proper enzymes to process the food you eat, it might take you 50 years to digest your breakfast.

• Catalysis is probably the most important function of proteins. • Catalysts that serve this function in organism are called enzymes. • With the exception of some recently discovered RNAs that have catalytic activity, all enzymes are proteins. • Enzymes are the most efficient specific catalysts known (increase rate reaction up to 10²º). • Nonenzymatic catalysts (up to 10²-104). • Enzymes are highly specific,even to the point of distinguishing stereoisomers of a given compound.

1)Enzymes are Effective Biological Catalysts

2)Difference between Kinetic and Thermodynamic Aspects of Reactions
• The rate of the reaction and its thermodynamic favorability are two different topics although so related. • The standard free energy change (ΔG°) is the difference between the energies of the reactants (initial state) and the energies of the products (final state).

• Enzymes like all catalysts speed up reaction but cannot alter the equilibrium constant of ΔG°. • Reaction rate depends on activation energy ΔG°‡ (energy input required to initiate the reaction). • Uncatalyzed reaction require more energy to get started,so its rate is slower than that of a catalyzed reaction.

•The Activation energy profile show the intermediate stages of a reaction between initial and final states. •The catalyst speed up a reaction changing the mechanism and thus lowering activation energy ΔG°‡(the amount of free energy required to bring the reactants to the transition state).

•The most important effect of catalyst is lowering ΔG°‡ , but not free energy change ΔG° . •Enzymes lowers ΔG°‡ needed for substrate molecule to reach the transition state. •Conversion of H2O2 to H2O+O2 provide an example of effect of catalysts on activation energy.

Temperature Effect :
• It increase the energy available to the reactants to reach the transition state, thus increasing the rate of chemical reaction. • More temperature increasing (>56° C ) enzyme denaturation. slowing down the reaction.

Enzymes are biological catalysts. They increase the rates of reactions by lowering the free energy of activation, but they don’t affect the thermodynamic aspects of reactions.

3) Description of Enzyme Kinetics in Mathematical Terms
A + B P Rate of disappearance of A = - Δ[A]/Δt = = = = B = - Δ[B]/Δt = = appearance of P = Δ[P]/Δt -Δ[A] -Δ[B] Δ[P] Rate=-------- =--------- =-------= k [A]f[B]g Δt Δt Δt K = rate constant, f & g must be determined experimentally. They are usually small whole numbers, such as 1 or 2 (0 in some cases).

The values of the exponents are related to the number of molecules involved in the detailed steps that constitute the mechanism: 1) A P, Rate=k [A]1 :first order reaction. 2) A+B C+D, Rate=k [A]1[B]1:2nd order reaction. 3) A B , Rate=k [A]0=k : zero order.
(enzyme-catalyzed reactions can exhibit zeroorder kinetics when the concentrations of reactants are so high that the enzyme is completely saturated with reactant molecules).

4)Binding of Substrates to Enzymes
• Enzyme binds to the substrate to form a complex ES; this leads to the transition-state species which then forms the product. • A substrate binds noncovalently to a small portion of the enzyme called active site (cleft-crevice) consisting of certain amino acids that are essential for enzymatic activity.

• The first step is the binding of substrate to the enzyme, which occurs because of highly specific interactions between the substrate and the side chains and backbone groups of the amino acids making up the active site. • Two important models have been developed to describe the binding process :

Lock-and-key model :assumes a high degree of similarity between the shape of the substrate and the geometry of the binding site on the enzyme.Substrate binds to a site whose shape complements its own, like a key in a lock.

Induced-fit model : The substrate binding induces a conformational change in the enzyme results in a complementary fit after the substrate is bound, taking in account the enzyme 3-dimensional shape flexibility.

•If binding was perfect attraction between E and S, which will cause ES complex to be lower on energy diagram than E+S at start. •Enzyme & substrate must bind to form ES complex before anything else can happen. •ES would be at such a low energy that the difference between ES and ES‡ would be very large.

• ES must attain the transition state EX‡. • This would slow down the rate of reaction. • Studies have shown that enzymes increase rate of reaction by lowering energy of transition state (EX‡ Induced fit model support this idea better than lock-and-key model).

• Catalysis occur after substrate is bound and transition state is formed. • In transition state, substrate is bound close and in correct orientation to atoms with which it is to react. Both effects (proximity and orientation) speed up reaction. • Bonds are broken and new bonds are formed. • Substrate is transformed into product. • Product is released from the enzyme, which can then catalyze more reaction. • Each enzyme has its unique mode of catalysis.

5)Examples of EnzymeCatalyzed Reactions
1) Chymotrypsin is a digestive enzyme : it catalyzes the hydrolysis of peptide bonds and ester bonds.

• Use p.nitrophenyl ester as convenient model system.

.It shows hyperbolic curve ( the rate of reaction rises as substrate concentration increases and a maximum rate is reached). .Like Myoglobin, it’s nonallosteric enzymes. .Michaelis-Menten equation can be used.

2) Aspartate transcarbamoylase (ATCase): .is an enzyme involved in the production of pyrimidines (UTPCTP) needed for the biosynthesis of DNA and RNA. .it shows sigmoidal curve.

• Allosteric protein,like Hemoglobin. • Michaelis–Menten equation cannot be used alone.

Allosteric proteins are the ones in which subtle changes at one site affect structure and function at another site.

6)The Michaelis-Menten Approach to Enzyme Kinetics
• Leonor Michaelis & Maud Menten (1913). k¹ k² E + S ES E + P k-1 k¹ : rate constant for the formation of ES, k-1 : rate constant of the reverse reaction, k² : rate constant for conversion of ES to P. Michaelis constant KM = K-1 + K2 / K1 Rate (Velocity) of an enzymatic reaction depends on the substrate concentration [S]. Vinit or V0 indicate initial velocity. Vmax is maximum velocity at infinite substrate concentration.

Michaelis constant : KM -the substrate concentration at Vmax/2. -the inverse measure of substrate affinity : High KM - Low affinity. Low KM - High affinity. Michaelis-Menten equation: Vmax [S] V=-------KM+ [S] [S]=KM V= Vmax/2

1)Linearizing the Michaelis-Menten Equation: • It’s easier to work with a straight line than a curve. • We can transform the hyperbola to straight by taking the reciprocals of both sides of the equation : Lineweaver-Burk double-reciprocal plot.
1 ----- = [S] kM 1 1 ------------ = ----------+---------- =-------x-----+------Vmax [S] Vmax[S] Vmax[S] Vmax [S] Vmax

kM + [S]

kM

y =

V

m

x + b

2) Turnover Number: • It’s a quantity equal to the catalytic constant k2 or kcat or kp . • It’s the number of moles of substrate that react to form product per mole of enzyme per unit time ( min. or sec.). • Assumes that the enzyme is fully saturated with substrate & the reaction is proceeding at the maximum rate. • Example: Catalase (convert hydrogen peroxide to water and oxygen) can transform 40 million moles of substrate to product every second.

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Inhibitor : a substance interferes with the action of an enzyme and slows the reaction rate. It can give a considerable amount of information about enzymatic reactions. A reversible inhibitor can bind to the enzyme and subsequently be released leaving the enzyme in its original condition. An irreversible inhibitor reacts with the enzyme to produce a protein that is not enzymatically active and from which the original enzyme cannot be regenerated. Uncompetitive inhibitors : an inhibitor can bind to the ES complex but not to free enzyme.

7)Response of Enzymatic Reactions to Inhibitors

Two major classes of reversible inhibitors can be ditinguished on the basis of the sites on the enzyme to which they bind : -Competitive inhibitor:The compound very similar in structure to the substrate,it binds to the active site and block access of the substrate to the active site. -Noncompetitive inhibitor: Bind to the enzyme at a site other than the active site, cause a change in the structure of the enzyme, especially around the active site, as a result of binding. The substrate is still able to bind to the active site, but the enzyme cannot catalyze the reaction when the inhibitor is bound to it.

1)Kinetics of Competitive Inhibition: -Competitive inhibitor: Vmax is unchanged, KM increase (x intercept change - y does not). -Competitive inhibition can be overcome by a sufficiently high substrate concentration. -Equation for enzymatic reaction becomes: +S EI E ES E + P : +I [E][I] KI=----[EI] Algebraically, in presence of inhibitor,KM increase [I] by factor 1 +----- . K

If we substitute kM(1+[I]/kI) for kM in equation :
1 ----V kM 1 1 =------- x ----- + ----Vmax [S] Vmax

1 kM [I] 1 1 ----- = -----(1+----) x---- + ----V Vmax k1 [S] Vmax

( y

=

m

X

+

b )

Slope kM/Vmax has increased by the factor (1+[I]/kI), it is now : kM [I] ---(1+---) Vmax kI
:

y intercept does not change.

2)Kinetics of Noncompetitive Inhibition: In presence and absence of noncompetitive inhibitor, slope and y intercept change, while x intercept don’t change. Vmax decrease, but kM remain the same. In presence of I, VImax has the form :

VImax =-----1+[I]/kI
1 kM [I] 1 1 [I] ----- = --------(1+----) x-----+------(1 + -----) V Vmax kI [S] Vmax KI ( y =

Vmax

m

x +

b )

The action of Enzymes can be inhibited reversibly or irreversibly. Irreversible inhibition normally involves formation or breaking of noncovalents bonds in the enzyme. In reversible inhibition,some substance can bind to the enzyme and subsequently be released. These reversible inhibitors can be divided into two major groups: competitive inhibitors that bind at the active site and prevent binding of substrate and noncompetitive inhibitors that bind at a site other than the active site, changing the structure of the enzyme and preventing catalysis.