LECTURE 4: Reaction Mechanisms and Inhibitors

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

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. [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 QUIZ (10 min) • How is enzyme specificity achieved/tercapai ? • Calculate Vmax & KM from the following data, and does the reaction obey Michaelis-Menten kinetics ?

8. ANSWERS • The enzyme specificity is achieved/tercapai through/siap the characteristic of active site/tempat • Vmax = 4.36695 KM = 2.2E-08 R2 = 0.999864, 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/pastienzymes in either a reversible/tetap or irreversible manner/cara, and thereby/dengan cara demikian block catalysis by that enzyme • Such/seperticompounds are called INHIBITORS and include/memasukkandrugs/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/sekutuwith 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 ituthe velocity/kecepatan of active enzyme is lowered in proportion/bagianto the concentration of the inhibitor and thus the effect is that of noncompetitive inhibition:

13. Examples of irreversible inhibitors include/memasukkandiisopropyl 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 inidescribed as kcat inhibition in that a latent/tersembunyiinhibitor 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/pemeriksaanfor active sites since they are not converted/dimasukkan from the latent/tersembunyito 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/meliputiequilibrium/kesetimbangan between the enzyme and the inhibitor, the equilibrium constant (Ki) being a measure/ukuran of the affinity/daya tarik - menarikof 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 thathigh 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 fallsmarkedly/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/berbedafrom competitive inhibitionin thatthe inhibitor can combine with ES, and S can combine with EIto form in both instances/hal, contohEIS.

28. This type of inhibition is not completely reversed/dibalikan by high substrate concentrationsince theclosed sequence/rangkaian will occur/terjadiregardless/tanpa memperhatikanof 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 langsungthat 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 balikoccur : terjadi

35. HOW TO SOLVE/memecahkanTHE 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. [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 SOAL • Diketahui suatu reaksi enzimatis tanpa dan dengan inhibitor dengan [I] = 2,2.104M. • Hitunglah KM dan Vmax tanpa dan dengan I serta KI