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3/17/2011
1
LECTURE 4:LECTURE 4:Reaction Mechanism and InhibitorsReaction Mechanism and Inhibitors
Kinetic data cannot unambiguously establish a reaction mechanism.Although a phenomenological description can be obtained the nature of the reaction intermediates remain indeterminate and other independent
measurements are needed.
Modes of Antimicrobial ActionModes of Antimicrobial ActionEnzyme Inhibition (Mechanism)
I
I
S
S
S I
I
I II
S
Competitive Non-competitive Uncompetitive
EE
Different siteCompete for
active siteInhibitor
Substrate
Car
toon
Gui
deEq
uatio
n and
Des
cripti
on
[II] binds to free [E] only,and competes with [S];increasing [S] overcomesInhibition by [II].
[II] binds to free [E] or [ES] complex; Increasing [S] cannot overcome [II] inhibition.
[II] binds to [ES] complex only, increasing [S] favorsthe inhibition by [II].
E + S→ES→E + P+II↓EII
←
↑
E + S→ES→E + P+ +II II↓ ↓EII+S→EIIS
←
↑ ↑
E + S→ES→E + P+II
↓EIIS
←
↑
EI
S
Juang RH (2004) BCbasics
LECTURE LAYOUTLECTURE LAYOUTREACTION MECHANISMSREACTION MECHANISMSA.A. Sequential ReactionsSequential ReactionsB.B. Random Random BisubstrateBisubstrate ReactionsReactionsC.C. PingPing--Pong ReactionsPong ReactionsINHIBITORSINHIBITORS1.1. Irreversible Irreversible 2.2. ReversibleReversible
Competitive inhibition,Competitive inhibition, Uncompetitive inhibition.Uncompetitive inhibition. Noncompetitive inhibition Noncompetitive inhibition
INTRODUCTIONINTRODUCTION
Thus far, we have considered only the simple Thus far, we have considered only the simple case of enzymes that act upon a single case of enzymes that act upon a single substrate, substrate, SS. This situation is not common. . This situation is not common. Usually, enzymes catalyze reactions in which Usually, enzymes catalyze reactions in which two (or even more) substrates take part.two (or even more) substrates take part.
Consider the case of an enzyme catalyzing a Consider the case of an enzyme catalyzing a reaction involving two substrates, reaction involving two substrates, AA and and BB, and , and yielding the products yielding the products PP and and QQ::
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Reaction MechanismsReaction MechanismsA: Sequential ReactionsA: Sequential Reactions All substrates must combine with enzyme All substrates must combine with enzyme
before reaction can occurbefore reaction can occur
Bisubstrate reactions
B. Random Bisubstrate Reactions Group transfer reactionsGroup transfer reactions One or more products One or more products
released before all released before all substrates addedsubstrates added
C. Ping-Pong Reactions
QUIZ (10 min)QUIZ (10 min)1.1. How is enzyme specificity achieved ?How is enzyme specificity achieved ?2.2. Calculate Vmax & KM from the following data, and dCalculate Vmax & KM from the following data, and does the oes the
reaction obey Michaelisreaction obey Michaelis--Menten kinetics ?Menten kinetics ?
[DNA]mol total
nucleotides/L
Free nucleotides in solution,V (pmol/L)
0 min 10 min1.0 x 10-5 0.05 5.11.0 x 10-6 0.04 4.51.0 x 10-7 0.06 3.21.0 x 10-8 0.04 1.41.0 x 10-9 0.04 0.23
ANSWERSANSWERS1.1. The enzyme specificity is achieved The enzyme specificity is achieved
through the characteristic of active sitethrough the characteristic of active site2.2. Vmax = Vmax = 4.366954.36695
KM = KM = 2.2E2.2E--0808RR22 = = 0.999864, so the reaction obeys 0.999864, so the reaction obeys MichaelisMichaelis--Menten kinetics Menten kinetics
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An important number of compounds have An important number of compounds have the the ability to combine with certain enzymesability to combine with certain enzymes in either a in either a reversible or irreversible manner, and thereby reversible or irreversible manner, and thereby block catalysis by that enzymeblock catalysis by that enzyme
Such compounds are called Such compounds are called INHIBITORS INHIBITORS and and include drugs, include drugs, antibioticsantibiotics, , poisonspoisons, anti , anti metabolitesmetabolites, as well as , as well as products of enzymic products of enzymic reactionsreactions
Two general classes of inhibitors are recognized ; Two general classes of inhibitors are recognized ; Irreversible Irreversible ReversibleReversible
INHIBITORS Inhibitors of Michaelis-Menten Enzymes
An irreversible inhibitor forms a covalent bond An irreversible inhibitor forms a covalent bond with a with a specific function, usually an amino acid residue, specific function, usually an amino acid residue, which may, in some manner, be associated with the which may, in some manner, be associated with the catalytic activity of the enzymecatalytic activity of the enzyme
There are many examples of enzyme inhibitors which There are many examples of enzyme inhibitors which covalently bind not at the active site, but physically covalently bind not at the active site, but physically block the active siteblock the active site
The inhibitor cannot be released by dilution or The inhibitor cannot be released by dilution or dialysisdialysis; ; kinetically, the concentration and hence the kinetically, the concentration and hence the velocity of active enzyme is lowered in proportion to velocity of active enzyme is lowered in proportion to the concentration of the inhibitor and thus the effect the concentration of the inhibitor and thus the effect is that of noncompetitive inhibition: is that of noncompetitive inhibition:
1. IRREVERSIBLE INHIBITORS
Irreversible Inhibition E + S ES E + P + I EI
KS
K I
Examples of irreversible inhibitors include;Examples of irreversible inhibitors include; diisopropyl fluorophosphate diisopropyl fluorophosphate which reacts irreversibly withwhich reacts irreversibly with serine serine
proteases, proteases, chymotrypsin, chymotrypsin, andand iodoacetate iodoacetate which reacts with essential sulfhydryl group of an which reacts with essential sulfhydryl group of an
enzyme such as enzyme such as triose phosphate dehydrogenasetriose phosphate dehydrogenase: :
E-SH+ICH2COOH E-SCH2COOH+HI
Reaction of the irreversible inhibitor diisopropylfluorophosphate (DFP) with a serine protease
Diisopropyl fluorophosphate
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A unique type of irreversible inhibition has been recently A unique type of irreversible inhibition has been recently described as kdescribed as kcatcat inhibition in that a latent inhibitor is inhibition in that a latent inhibitor is activated to an active inhibitor by binding to the active activated to an active inhibitor by binding to the active site of the enzyme.site of the enzyme.
The newly generated inhibitor now reacts chemically with The newly generated inhibitor now reacts chemically with the enzyme leading to its irreversible inhibitionthe enzyme leading to its irreversible inhibition
These inhibitors have great potential as These inhibitors have great potential as drugsdrugs in highly in highly specific probes for active sites since they are not specific probes for active sites since they are not converted from the latent to the active form except by converted from the latent to the active form except by their specific target enzymestheir specific target enzymes
An excellent example is the inhibition of An excellent example is the inhibition of DD--33--hydroxyl hydroxyl decanoyl ACP clehydrasedecanoyl ACP clehydrase (of E. coli) by the latent inhibitor (of E. coli) by the latent inhibitor 33--decynoyldecynoyl--NN--acetyl cystamineacetyl cystamine according to the following according to the following sequences of events:sequences of events:
2. REVERSIBLE INHIBITION2. REVERSIBLE INHIBITION As the term implies, this type of inhibition involves As the term implies, this type of inhibition involves
equilibrium between the enzyme and the inhibitor, equilibrium between the enzyme and the inhibitor, the equilibrium constant (Ki) being a measure of the equilibrium constant (Ki) being a measure of the affinity of the inhibitor for the enzyme. the affinity of the inhibitor for the enzyme.
Three distinct types of reversible inhibition are Three distinct types of reversible inhibition are known; known; Competitive inhibition,Competitive inhibition, Uncompetitive inhibition Uncompetitive inhibition Noncompetitive inhibition Noncompetitive inhibition
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A. Competitive InhibitionA. Competitive Inhibition Compounds that may or may not be structurally Compounds that may or may not be structurally
related to the natural substrate combine related to the natural substrate combine reversibly with the enzyme at or near the active reversibly with the enzyme at or near the active sitesite
The inhibitor and the substrate therefore The inhibitor and the substrate therefore compete for the same site according to the compete for the same site according to the reaction: reaction:
]S[K
]I[1K
]S[VV
IM
max
Competitive Inhibition
Succinate Glutarate Malonate Oxalate
Succinate Dehydrogenase
Substrate Competitive InhibitorProduct
Adapted from Kleinsmith & Kish (1995) Principles of Cell and Molecular Biology (2e) p.49
C-OO-
C-H
C-H
C-OO-
C-OO-
H-C-H
H-C-H
C-OO-
C-OO-
H-C-H
H-C-H
H-C-H
C-OO-
C-OO-
C-OO-
C-OO-
H-C-H
C-OO-
ES and EI complexes are formed, but EIS complexes are never produced. One can conclude that high concentrations of substrate will overcome the inhibition by causing the reaction sequence to swing to the right. The velocity of reaction can be calculated by the following equation
C o m p etitiv e in h ib ito r
-I
+I
-1/KM -1/[K M(1+1/KI)]
1/V
1/S
Among other enzymes that may undergo competitive Among other enzymes that may undergo competitive inhibition (Table 1) is inhibition (Table 1) is succinic dehydrogenasesuccinic dehydrogenase, which , which readily oxidizes succinic acid to fumaric acid. readily oxidizes succinic acid to fumaric acid.
If increasing concentrations of malonic acid, which closely If increasing concentrations of malonic acid, which closely resembles succinic acid in structure, are added, however, resembles succinic acid in structure, are added, however, succinic dehydrogenase activity falls succinic dehydrogenase activity falls markedly. This markedly. This inhibition can now be reversed by increasing in turn the inhibition can now be reversed by increasing in turn the concentration of the substrate succinic acid. concentration of the substrate succinic acid.
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HIV protease vs Aspartyl protease
Asymmetricmonomer
↓ HIV protease HIV protease (homodimer)HIV Protease inhibitor is used in treating AIDS
Symmetricdimer
Asp
subunit 2
↑Aspartyl protease (monomer)
subunit 1Asp
domain 1 domain 2
Asp Asp
Juang RH (2004) BCbasics
B. Uncompetitive InhibitionB. Uncompetitive Inhibition Compounds that combine only with the ES complex, not Compounds that combine only with the ES complex, not
with the free enzyme, are called uncompetitive inhibitors. with the free enzyme, are called uncompetitive inhibitors. The inhibition is not overcome by high substrate The inhibition is not overcome by high substrate concentrations. concentrations.
The structure of the protease is The structure of the protease is shown by the red, blue and yellow shown by the red, blue and yellow ribbons. The inhibitor is shown as ribbons. The inhibitor is shown as the smaller ballthe smaller ball--andand--stick structure stick structure near the centre. Created from PDBnear the centre. Created from PDB
Human immunodeficiency virusScanning electron micrograph of HIV-1 (in green) budding from cultured lymphocyte. Multiple round bumps on cell surface represent sites of assembly and budding of virions
Peptide-based protease inhibitor ritonavir
HIV protease HIV protease in a complex with the protease inhibitor in a complex with the protease inhibitor ritonavirritonavir
KKMM value is consistently smaller than the Kvalue is consistently smaller than the KMM value value of the uninhibited reaction which implies that S is of the uninhibited reaction which implies that S is more effectively bound to the enzyme in the more effectively bound to the enzyme in the presence of the inhibitor. presence of the inhibitor.
The equation used to calculate the velocity of the The equation used to calculate the velocity of the noncompetitive inhibition is as follows noncompetitive inhibition is as follows
IM
max
K]I[1]S[K
]S[VV
Uncompetitive inhibitor
-I
+I
-1/V m ax
(1+[I]/K I)/Vm ax
-(1+[I]/K I)/K M
-1/K M
1/S
1/V
C. Noncompetitive InhibitionC. Noncompetitive Inhibition Compounds that reversibly bind with either the enzyme or Compounds that reversibly bind with either the enzyme or
the enzyme substrate complex are designated as the enzyme substrate complex are designated as noncompetitive inhibitorsnoncompetitive inhibitors
Noncompetitive inhibition therefore differs from Noncompetitive inhibition therefore differs from competitive inhibition in that the inhibitor can combine competitive inhibition in that the inhibitor can combine with ES, and S can combine with EI to form in both with ES, and S can combine with EI to form in both instances EIS. instances EIS.
This type of inhibition is not completely reversed by high This type of inhibition is not completely reversed by high substrate concentration substrate concentration since thesince the closed sequence will closed sequence will occur regardless of the substrate concentrationoccur regardless of the substrate concentration
Since the inhibitor binding site is not identical to nor does Since the inhibitor binding site is not identical to nor does it modify the active site directly, the Kit modify the active site directly, the KMM is not altered. is not altered.
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IM
max
K]I[1]S[K
]S[VV
Noncompetitive
-I
+I
-1/Vmax
(1+[I]/KI)/Vmax
1/V
1/S
FEEDBACK FEEDBACK INHIBITIONINHIBITION
The switch: Allosteric inhibitionThe switch: Allosteric inhibition
Allosteric means Allosteric means “other site”“other site”
E
Active site
Allosteric site
© 2008 Paul Billiet ODWS
Switching offSwitching off
One site fits the One site fits the substrate like other substrate like other enzymesenzymes
The other site fits The other site fits an inhibitor an inhibitor moleculemolecule
Inhibitor fits into allosteric site
Substratecannot fit into the active site
Inhibitor molecule
© 2008 Paul Billiet ODWS
These enzymes have These enzymes have two receptor sitestwo receptor sites
HOW TO SOLVE THE EQUATIONSHOW TO SOLVE THE EQUATIONS
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1. 1. CompetitiveCompetitive inhibitorinhibitor
y =1/V; x = 1/[s]y =1/V; x = 1/[s] a = 1/a = 1/VmaxVmax b = Kb = KMM(1+[I]/K(1+[I]/KII)/)/VmaxVmax
]S[K
]I[1K
]S[VV
IM
max
maxV1
]S[1
VK
]I[1K
V1
max
IM
2. Uncompetitive 2. Uncompetitive
y =1/V; x = 1/[s]y =1/V; x = 1/[s] a = (1+[I]/Ka = (1+[I]/KII)/Vmax)/Vmax b = Kb = KMM/Vmax /Vmax
IM
max
K]I[1]S[K
]S[VV
maxVK
]I[1
]S[1
maxVK
V1 IM
3. 3. Noncompetitive InhibitionNoncompetitive Inhibition
y =1/V; x = 1/[s]y =1/V; x = 1/[s] a = (1+[I]/Ka = (1+[I]/KII)/Vmax)/Vmax b = Kb = KMM(1+[I]/K(1+[I]/KII)/Vmax )/Vmax
IM
max
K]I[1]S[K
]S[VV
maxVK
]I[1
]S[1
maxVK
]I[1K
V1 II
M
SOALSOAL
Diketahui suatu Diketahui suatu reaksi enzimatis reaksi enzimatis tanpa dan dengan tanpa dan dengan inhibitor dengan [I] = inhibitor dengan [I] = 2,2.102,2.1044M.M.
Hitunglah KM dan Hitunglah KM dan Vmax tanpa dan Vmax tanpa dan dengan I serta Kdengan I serta KII
[S] V(-I) V(+I)
1*10-4 28 17
1.5*10-4 36 23
2.0*10-4 43 29
5*10-4 65 50
7.5*10-4 74 61
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