Documente Academic
Documente Profesional
Documente Cultură
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
simplifies to:
8/24/2012
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 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]
8/24/2012
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
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]
8/24/2012
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
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]
8/24/2012
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
KM
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)
8/24/2012
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
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
v0/[S]
With inhibitor v0
8/24/2012
[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]
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
8/24/2012
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
8/24/2012
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]
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
8/24/2012
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
1/[A]
Vmax
10
8/24/2012
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]
11
8/24/2012
Log Vmax
pH
12
8/24/2012
13