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Enzyme Inhibition

Consider the following general scheme for enzyme inhibition: Ks k2 E + S ES E + P +I +I KEI KESI Ks EI + S ESI

Assuming that all the enzymecontaining complexes are in equilibrium with each other, the general equation can be given by:

where: [S] and [I] :substrate and inhibitor concentrations, respectively. KS, KESI, KEI : dissociation constants The equation can be simplified by making certain assumptions.

1. Competitive inhibition If in the general equation, KESI = , i.e. the ES complex cannot combine with I, nor the EI complex with S. Ks k2 E + S ES E + P +I +I KEI KESI Ks EI + S ESI

The general equation

simplifies to:

This is known as competitive inhibition.

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A molecule that resembles the substrate in structure may occupy the catalytic site but may be completely unreactive. By occupying the active site, the molecule acts as competitive inhibitor, in preventing normal substrates from being catalyzed The effect is to have the inhibitor pull some of the enzyme into the form of the EI complex E + S ESI E + I EI

Competitive inhibitors bind reversibly to the active site. The inhibition can be reversed by: Diluting the inhibitor; or Adding a large excess of substrate

Effect of competitive inhibition: No effect on Vmax increase KM

Vmax No inhibitor v0 Vmax With inhibitor

Effect on LineweaverBurk plot no change in yintercept increase in slope decrease in xintercept 1/KM 1/v0 with inhibitor no inhibitor

1/KM

1/Vmax

KM = KM 1 + [I]/KEI

KM

KM

[S]

1/[S]

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From the previous plot KM = KM {1 + ([I]/KEI) We get, (KM/KM) = 1 + ([I]/KEI) Therefore, for the plot of (KM/KM) versus [I] slope = 1/KEI and yintercept = 1

Effect on EadieHofstee Plot no change in xintercept decrease in slope by 1/{1+[I]/KEI} no inhibitor Slope = - 1/KM

v0/[S] Slope = - 1/KM with inhibitor

Vmax

v0

Effect on Hanes Plot no change in slope increase in xintercept and y intercept with inhibitor no inhibitor KM KM Slope = 1/Vmax

[S]/v0

2. Noncompetitive inhibition If in the general equation, KESI = KEI, i.e. the binding of S to the enzyme does not affect the binding of I, then

[S]

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KM remains unaffected Vmax is decreased by a factor 1/{1 + ([I]/KEI)} Net result : decrease in KM, but unchanged Vmax equivalent to conversion of enzyme to inactive form e.g. Diisopropylfluorophosphate as noncompetitive inhibitor of chymotrypsin inactivates the enzyme by reacting with an essential serine group

Effect of noncompetitive inhibition: no change in KM decrease in Vmax

Vmax No inhibitor Vmax With inhibitor

v0 Vmax Vmax

KM = KM

[S]

Effect on LineweaverBurk plot increase in slope by a factor of {1 + ([I]/KEI)} increase in yintercept by a 1/Vmax factor {1 + ([I]/KEI)}
with inhibitor 1/v0 no inhibitor 1/KM 1/Vmax = {1 + ([I]/KEI)}/Vmax 1/Vmax

Since 1/Vmax = {1 + ([I]/KEI)}Vmax (Vmax/Vmax)= 1 + [I]/KEI Hence for the plot of (Vmax/Vmax) versus [I], Slope = 1/KEI yint = 1.0

1/[S]

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Effect on EadieHofstee Plot no change in slope decrease in x intercept by a factor 1/{1 + ([I]/KEI)}
V max = Vmax/{1 + ([I]/KEI)}
v0/[S] Vmax

Effect on Hanes Plot no change in xintercept increase in slope by factor {1 + ([I]/KEI)}

[S]/v0 with inhibitor Slope = 1/Vmax = {1 + ([I]/KEI)}/Vmax no inhibitor Slope = 1/Vmax

no inhibitor Slope = - 1/KM

KM

With inhibitor v0 [S]

3. Uncompetitive inhibition If in the general equation, KEI = , i.e. E cannot combine with I, then

Both KM and Vmax are affected Both KM and Vmax are decreased by a factor of {1 + ([I]/KESI)

Extremely rare in onesubstrate reactions (e.g. inhibition of rat intestinal alkaline phosphatase) but more often found in multisubstrate reactions (e.g. S adenosylmethionine towards ATP in the reaction catalyzed by yeast S adenosylmethionine synthase)

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Effect of uncompetitive inhibition: decrease in Vmax decrease in KM

Vmax No inhibitor

Effect on LineweaverBurk plot No change in slope increase in yintercept by a factor of {1 + ([I]/KESI)} increase in xintercept by a factor {1 + ([I]/KESI)}
with inhibitor no inhibitor

1/Vmax 1/v0

v0 Vmax

Vmax With inhibitor

1/KM 1/KM 1/Vmax Vmax = Vmax /{1 + ([I]/KESI)} KM = KM/{1 + ([I]/KESI)}

Vmax

KM

KM

[S]

1/[S]

From the Lineweaver Burk plot, Vmax = Vmax /{1 + ([I]/KESI)} and (Vmax/Vmax) = 1 + ([I]/KESI Hence, if (Vmax/Vmax) is plotted vs [I] Slope = 1/KESI and yint = 1.0 Alternatively, KM = KM/{1 + ([I]/KESI)} (KM/KM) = 1 + ([I]/KESI) Therefore, for the plot of (KM/KM) vs [I], Slope = 1/KESI

Effect on EadieHofstee Plot no change in yintercept decrease in xintercept by a factor 1/{1 + ([I]/KESI)} increase in slope by a Slope = 1/KM factor 1/{1 + ([I]/K )} ESI
no inhibitor

V max = Vmax/{1 + ([I]/KESI)}

Vmax Slope = 1/KM = {1+ ([I]/KESI}/KM

v0/[S]

With inhibitor v0

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Effect on Hanes Plot

[S]/v0

no change in yintercept increase in slope by factor {1 + ([I]/KESI)} decrease in xint by factor {1 + [I]/KESI}
Slope = 1/Vmax = {1 + ([I]/KESI)}/Vmax no inhibitor

The effect of choline on the reaction catalyzed by insect acetylcholinesterase was studied with the following results:
[choline] mM 0 20 40 0.10 [Acetylcholine] mM 0.15 0.25 0.40 0.70

with inhibitor
KM = KM/{1 + [I]/KESI}

KM

Slope = 1/Vmax

(relative velocity arbitrary units) 28.5 37.5 50.0 61.5 73.7 11.9 15.6 20.9 25.7 30.7 7.5 9.9 13.2 16.2 19.4

[S]

What type of inhibition is being observed? What is the value of KEI/KESI ?

1/[acetylcholine] 6.667 4.000 2.500 1.429 [Choline] 1/velocity 0 0.035088 0.026667 0.02 0.01626 0.013569 20 mM 0.084034 0.064103 0.047847 0.038911 0.032573 40 mM 0.133333 0.10101 0.075758 0.061728 0.051546 10.000 y-int slope No I 0.009975 0.002509 20 mM 0.023907 0.006015 44 mM 0.037791 0.009531

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Two substrate kinetics Category Hydrolases Lyases Isomerases Oxidoreductases Transferases Ligases Type Reaction Catalyzed 1S Hydrolysis 1S Elimination of a group to form a double bond 1 S Isomerization 2 S Redox, one S is reduced at the expense of the other 2 S Group transfer 2 S Joining together of 2 molecules at the expense of ATP, or some other energy source

2 Substrate Mechanisms 1. Those involving a ternary complex Proceed via EAB and EPQ types of complexes (A and B are the reactants, P and Q are the products) E + A + B EAB EPQ E + P + Q a. Ternary complex is formed in ordered manner E + A EA EA + B EAB (but E + B x EB) b. Ternary complex is formed in random manner E + A EA EA + B EAB or E + B EB EB + A EAB

2. Those not involving a ternary complex a. Reactions in which the first product is formed before the second substrate is bound involve a modification of the enzyme and are known as enzyme substitution or ping pong mechanisms E + A E + P E + B E + Q b. Reactions in which a ternary complex is presumably formed but its breakdown to yield the first product is very fast so that the ternary complex is kinetically insignificant TheorellChance mechanism e.g. oxidation of ethanol catalyzed by horseliver alcohol dehydrogenase

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Kinetic equation for 2S reactions Velocity of the overall reaction is equal to the concentration of the complex preceding enzyme regeneration multiplied by the rate constant for the step which regenerates the enzyme For the ternary complex mechanism: Vmax [ A ] [B] v0= KA KB + KB [A] + KA[B] + [A][B] For the pingpong mechanism v0 = Vmax KB [A] + KA[B] + [A][B]

Vmax = maximum velocity at saturating levels of A and B KA, KB : Michaelis Menten contants for each substrate in the presence of saturating concentrations of the other substrate Ex. For the ternary complex mechanism, if both numerator and denominator are divided by [B], then [B] is set to , the equation reduces to v0 = Vmax [A] KA + [A]

This shows that KA is the constant for A at saturating levels of B

If the equation is divided by [A] and at saturating levels of A, vo =

Vmax [B] KB [A] + [A][B]

KA, KA, KB represent combinations of rate constants of individual steps in the reactions; the meaning varies according to the type of mechanism For random order ternary complex mechanisms EA KA E KB EB Where: KB = KAKB/KA KB EAB E + Products KA

where: KB = MichaelisMenten constant for B at saturating levels of A

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Derivation of Kinetic Parameters

For the ternary complex mechanism 1 v0 = KA [A] + KB + [B] KAKB [A][B] 1 Vmax

Secondary plots of the slope of the primary plot vesus 1/[B] Slope = (1/Vmax) {KA + (KAKB)/[B]}

Therefore for 1/v0 vs. 1/[A] at constant [B] Slope = (1/Vmax) {KA + (KAKB)/[B]} y int = 1/Vmax ( 1 + KB/[B]) [B] increasing 1/v0

Slope = (KA KB)/Vmax slope

yint = KA/Vmax 1/[B]

1/[A]

Secondary plots of the yintercepts of the primary plots vs 1/[B]

If a similar procedure is adopted for the pingpong mechanism, KA + [A] KB [B]

1

1/vo = 1 + Slope = KB/Vmax yintercept Yintercept = 1/Vmax 1/[B]

Vmax

Hence, for a plot of 1/v vs 1/[A] at constant [B]

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[B] increasing Slope = KA/Vmax

Characteristics of ping pong mechanism: Slope is independent of [B] y int decreases as [B] increases Secondary plot is obtained from plot of yint vs [B] slope = KB/Vmax Y int y int = 1/Vmax

1/vo
yint = 1 + (KB/[B] (1/Vmax)

1/[A]
1/[B]

Distinction between Various Mechanisms

To confirm a ping pong mechanism, the following should be detected: 1. Occurrence of half reactions; i.e., formation of the first product in the absence of the second substrate; 2. The formation of a modified enzyme on incubation with the first substrate.

Factors to be considered to distinguish between ordered and random mechanism:

1. Product inhibition pattern - the type of inhibition shown by the products (P/Q) towards the substrates (A/B) can be used to exclude a likely mechanism 2. Substrate binding - whether E binds the second substrate or not random: A & B both bind to the enzyme ordered: the 2nd substrate cannot bind to E in the absence of the 1st substrate

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PreSteady State Kinetics

When [E] and [S] are nearly equal 3. Isotope exchange at equilibrium - involves measuring the concentration of substrates (and products) on the rate of exchange of an isotope between S and P when the reaction is at equilibrium Information available: 1. Participation of complexes and intermediates in the reaction pathway; rate constants 2. Amount of enzyme undergoing the reaction can be determined from the amount of intermediate accumulated (or product released as the intermediate is formed) if the breakdown of the intermediate is very slow

Effect of pH on enzymecatalyzed reactions

1. Unfolding and consequent inactivation of the protein outside the pH range 2. Changes in the position of equilibrium of a reaction if H+ appears as a reactant or product in the over all reaction 3. Ionization of groups(s) in the substrate 4. Changes in the ionization state of amino acid side chains in the enzyme

If log Vmax is plotted vs pH

Log Vmax

pH

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