LECTURE 4:Reaction

Mechanisms and Inhibitors

Reaction MechanismsA: Sequential/rangkaian Reactions• All substrates must

combine/digabungkan with enzyme before reaction can occur/terjadi

Bisubstrate reactions

B. Random Bisubstrate Reactions

C. Ping-Pong Reactions• Group transfer reactions• One or more products

released/dibebaskan before all substrates added

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.

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

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

• 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

• 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

Irreversible Inhibition E + S ES E + P + I EI

KS

KI

• 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.

• 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:

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.

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

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

C om p etitive in h ib itor

-I

+I

-1/KM -1/[KM(1+1/KI)]

1/V

1/S

• 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.

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.

• 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

N oncom petitive

-I

+ I

-1/V m ax

(1+ [I]/K I)/V m ax

1/V

1/S

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

• 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

Uncompetitive inhibitor

-I

+I

-1/Vmax

(1+[I]/KI)/Vmax

-(1+[I]/KI)/KM

-1/KM

1/S

1/V

FEEDBACK INHIBITION/perintang pengaruh arus balik

occur : terjadi

HOW TO SOLVE/memecahkan THE EQUATIONS

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

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

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

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