1. LECTURE 4: Reaction Mechanisms and Inhibitors

2. Reaction Mechanisms A: Sequential/rangkaian Reactions
All substrates must combine/digabungkan with enzyme before reaction can occur/terjadi

3. Bisubstrate reactions
Single displacement reactions

4. B. Random Bisubstrate Reactions

5. C. Ping-Pong Reactions Group transfer reactions
One or more products released/dibebaskan before all substrates added

6. 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.

7. mol total nucleotides/L Free nucleotides in solution,
QUIZ (10 min)
How is enzyme specificity achieved/tercapai ?
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
1.4
1.0 x 10-9
0.23

8. ANSWERS
The enzyme specificity is achieved/tercapai through/siap the characteristic of active site/tempat
Vmax =
KM = 2.2E-08
R2 = , so the reaction obeys/menuruti Michaelis-Menten kinetics

9. INHIBITORS/penghalang
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

11. 1. IRREVERSIBLE INHIBITORS
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:

13. 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.

14. 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:

19. 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.

21. 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:

22. 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

23. 1/V
1/S

25. 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.

27. 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 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.

28. 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

29. 1/V
1/S

31. 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.

32. 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

33. 1/V
1/S

34. FEEDBACK INHIBITION/perintang pengaruh arus balik occur : terjadi

35. HOW TO SOLVE/memecahkan THE EQUATIONS

36. 1. Competitive inhibitor
y =1/V; x = 1/[s]
a = 1/Vmax
b = KM(1+[I]/KI)/Vmax

37. 2. Noncompetitive Inhibition
y =1/V; x = 1/[s]
a = (1+[I]/KI)/Vmax
b = KM(1+[I]/KI)/Vmax

38. 3. Uncompetitive
y =1/V; x = 1/[s]
a = (1+[I]/KI)/Vmax
b = KM/Vmax

39. SOAL
[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
Diketahui suatu reaksi enzimatis tanpa dan dengan inhibitor dengan [I] = 2,2.104M.
Hitunglah KM dan Vmax tanpa dan dengan I serta KI