LECTURE 4: Reaction Mechanisms and Inhibitors
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Transcript of LECTURE 4: Reaction Mechanisms and Inhibitors
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LECTURE 4:Reaction
Mechanisms and Inhibitors
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Reaction MechanismsA: Sequential/rangkaian Reactions• All substrates must
combine/digabungkan with enzyme before reaction can occur/terjadi
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Bisubstrate reactions
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B. Random Bisubstrate Reactions
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C. Ping-Pong Reactions• Group transfer reactions• One or more products
released/dibebaskan before all substrates added
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Kinetic data cannot unambiguously establish/membuat a reaction mechanism.
Although a phenomenological description can be obtained/dihasilkan the nature of the reaction intermediates remain/sisa indeterminate and other independent measurements/ukuran are needed.
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QUIZ (10 min)1. How is enzyme specificity achieved/tercapai ?2. Calculate Vmax & KM from the following data, and
does the reaction obey Michaelis-Menten kinetics ?
[DNA]mol total
nucleotides/L
Free nucleotides in solution,V (pmol/L)
0 min 10 min
1.0 x 10-5 0.05 5.1
1.0 x 10-6 0.04 4.5
1.0 x 10-7 0.06 3.2
1.0 x 10-8 0.04 1.4
1.0 x 10-9 0.04 0.23
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ANSWERS
1. The enzyme specificity is achieved/tercapai through/siap the characteristic of active site/tempat
2. Vmax = 4.36695 KM = 2.2E-08 R2 = 0.999864, so the
reaction obeys/menuruti Michaelis-Menten kinetics
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• An important number of compounds/senyawa have the ability/kemampuan to combine with certain/pasti enzymes in either a reversible/tetap or irreversible manner/cara, and thereby/dengan cara demikian block catalysis by that enzyme
• Such/seperti compounds are called INHIBITORS and include/memasukkan drugs/obat bius, antibiotics, poisons/racun, anti metabolites, as well as products of enzymic reactions
• Two general classes of inhibitors are recognized/yang diakui ; – Irreversible – Reversible
INHIBITORS/penghalang
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• An irreversible inhibitor forms/bentuk a covalent bond with a specific function, usually an amino acid residue/sisa, which may, in some manner/cara, be associated/sekutu with the catalytic activity of the enzyme
• There are many examples of enzyme inhibitors which covalently bind not at the active site, but physically block the active site
• The inhibitor cannot be released/dilepaskan by dilution/mencairkan or dialysis; kinetically/dengan gerakan, the concentration and hence/karena itu the velocity/kecepatan of active enzyme is lowered in proportion/bagian to the concentration of the inhibitor and thus the effect is that of noncompetitive inhibition:
1. IRREVERSIBLE INHIBITORS
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Irreversible Inhibition E + S ES E + P + I EI
KS
KI
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• Examples of irreversible inhibitors include/memasukkan diisopropyl fluorophosphate, which reacts irreversibly with serine proteases, chymotrypsin and iodoacetate which reacts with essential/perlu sulfhydryl group of an enzyme such/seperti as triose phosphate dehydrogenase:
E-SH+ICH2COOH E-SCH2COOH+HI
• A unique type of irreversible inhibition has been recently/baru2 ini described as kcat inhibition in that a latent/tersembunyi inhibitor is activated to an active inhibitor by binding to the active site of the enzyme.
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• The newly generated inhibitor now reacts chemically with the enzyme leading to its irreversible inhibition
• These inhibitors have great potential as drugs in highly specific probes/pemeriksaan for active sites since they are not converted/dimasukkan from the latent/tersembunyi to the active form except by their specific target enzymes
• An excellent example is the inhibition of D‑3‑hydroxyl decanoyl ACP clehydrase (of E. coli) by the latent inhibitor 3‑decynoyl‑N‑acetyl cystamine according/persetujuan to the following sequences/rangkaian of events:
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2. REVERSIBLE INHIBITION• As the term/istilah implies/menyatakan secara tidak
langsung, this type of inhibition involves/meliputi equilibrium/kesetimbangan between the enzyme and the inhibitor, the equilibrium constant (Ki) being a measure/ukuran of the affinity/daya tarik - menarik of the inhibitor for the enzyme.
• Three distinct/jelas types of reversible inhibition are known; – Competitive inhibition,– Noncompetitive inhibition – Uncompetitive inhibition.
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A. Competitive Inhibition
• Compounds that may or may not be structurally related to the natural substrate combine reversibly with the enzyme at or near the active site
• The inhibitor and the substrate therefore compete for the same site according to the reaction:
]S[K
]I[1K
]S[VV
IM
max
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Competitive Inhibition E + S ES E + P + I EI
KS
KI
ES and EI complexes are formed/dibentuk, but EIS complexes are never produced. One can conclude/mengakhiri that high concentrations of substrate will overcome the inhibition by causing the reaction sequence/rangkaian to swing/perjalanan to the right. The velocity/kecepatan of reaction can be calculated by the following equation/persamaan
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C om p etitive in h ib itor
-I
+I
-1/KM -1/[KM(1+1/KI)]
1/V
1/S
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• Among/diantara other enzymes that may undergo competitive inhibition (Table 1) is succinic dehydrogenase, which readily oxidizes succinic acid to fumaric acid.
• If increasing/penambahan concentrations of malonic acid, which closely resembles/mirip succinic acid in structure, are added, however, succinic dehydrogenase activity falls markedly/nyata. This inhibition can now be reversed/dikembalikan by increasing in turn the concentration of the substrate succinic acid.
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B. Noncompetitive Inhibition • Compounds that reversibly bind with either
the enzyme or the enzyme substrate complex are designated/dicalonkan as noncompetitive inhibitors and the following reactions describe these events: Noncompetitive Inhibition
E + S ES E + P + + I I EI + S EIS
KS
KI KI
KS
• Noncompetitive inhibition therefore differs/berbeda from competitive inhibition in that the inhibitor can combine with ES, and S can combine with EI to form in both instances/hal, contoh EIS.
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• This type of inhibition is not completely reversed/dibalikan by high substrate concentration since the closed sequence/rangkaian will occur/terjadi regardless/tanpa memperhatikan of the substrate concentration.
• Since the inhibitor binding site is not identical to nor does it modify the active site directly, the KM is not altered/diubah. The equation/persamaan used to calculate the velocity/kecepatan of the noncompetitive inhibition is as follows
IM
max
K
]I[1]S[K
]S[VV
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N oncom petitive
-I
+ I
-1/V m ax
(1+ [I]/K I)/V m ax
1/V
1/S
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C. Uncompetitive Inhibition • Compounds that combine only with the
ES complex but not with the free enzyme are called uncompetitive inhibitors. The inhibition is not overcome by high substrate concentrations.
Uncompetitive Inhibition E + S ES E + P + I EIS
KS
KI
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• Interestingly the KM value is consistently smaller than the KM value of the uninhibited reaction, which implies/secara tidak langsung that S is more effectively bound to the enzyme in the presence/kehadiran of the inhibitor.
• The equation/persamaan used to calculate the velocity/kecepatan of the noncompetitive inhibition is as follows
IM
max
K]I[
1]S[K
]S[VV
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Uncompetitive inhibitor
-I
+I
-1/Vmax
(1+[I]/KI)/Vmax
-(1+[I]/KI)/KM
-1/KM
1/S
1/V
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FEEDBACK INHIBITION/perintang pengaruh arus balik
occur : terjadi
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HOW TO SOLVE/memecahkan THE EQUATIONS
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1. Competitive inhibitor
•y =1/V; x = 1/[s]•a = 1/Vmax•b = KM(1+[I]/KI)/Vmax
]S[K
]I[1K
]S[VV
IM
max
maxV
1
]S[
1
V
K
]I[1K
V
1
max
IM
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2. Noncompetitive Inhibition
•y =1/V; x = 1/[s]•a = (1+[I]/KI)/Vmax
•b = KM(1+[I]/KI)/Vmax
IM
max
K
]I[1]S[K
]S[VV
maxV
K
]I[1
]S[
1
maxV
K
]I[1K
V
1 IIM
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3. Uncompetitive
•y =1/V; x = 1/[s]•a = (1+[I]/KI)/Vmax
•b = KM/Vmax
IM
max
K]I[
1]S[K
]S[VV
maxV
K]I[
1
]S[1
maxV
K
V1 IM
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SOAL
• Diketahui suatu reaksi enzimatis tanpa dan dengan inhibitor dengan [I] = 2,2.104M.
• Hitunglah KM dan Vmax tanpa dan dengan I serta KI
[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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