ENZIM

(LANJUTAN)

17 OKTOBER 2011

SEJARAH PENEMUAN ENZIM DAN ETIMOLOGI ENZIM

Contoh-contoh nomor EC untuk enzim

Esterase = Peptidase = Amilase = Fosfatase = Lipase = Dehidrogenase = Oksidase =

peroksidase = reduktase = xantin oksidase = as.amino oksidase = glisin oksidase =

OXIDOREDUCTASE:
Oxidizing or reducing a compound/ Transfering electron to or from a compound
C
CH2 CH2 C OH O

O
SUCCINATE DEHYDROGENASE

C
CH CH

FAD

FADH2

OH

SUCCINATE

FUMARATE

HO

HO

HYDROLASE:
Lysing a compound with the help of water
CH2OH
C H H C OH OH C H O CH2OH O H C OH CH2OH C H H C OH OH C H O H C OH

C H H H C C OH H O C C H OH H OH

H C OH

H C OH

H2O

TRANSFERASE:
Transfering a functional group across molecules

COOH
COOH
+H N 3

COOH
COOH ASPARTATE TRANSAMINASE C O
+H 3N

C CH2

C CH2 CH2

CH2 CH2 COOH

CH2

COOH L-ASPARTATE

a-KETO GLUTARATE

COOH L-GLUTAMATE

COOH OXALOACETATE

ISOMERASE:
Transfering a functional group within a molecule
H C H HO H H C C C O OH H OH OH O PO4 PHOSPHOGLUCO ISOMERASE HO H H CH2OH C C C O H OH OH O PO4

C
CH2

C
CH2

D-GLUCOSE 6-PHOSPHATE

D-FRUCTOSE 6-PHOSPHATE

LYASE:
Cleaving an organic bond within a molecule
COOH
HO CH HC CH2 COOH ISOCITRATE COOH ISOCITRATE LYASE COOH CH2 CH2 COOH SUCCINATE GLYOXILATE COOH HC O

LIGASE:
Formation of new organic bonds
OH HO P O C H O CH2

O
C H

HO

OH P O O

OH P O O

H C OH OH P O O

H C OH

+
O C H

LIGASE

CH2

C H

H C OH

H C OH

LIGASE:
Formation of new organic bonds
OH HO P O C H H C O P O O HO O CH2

O
C H

CH2

O C H

C H

H C OH

H C OH

H C OH

HO

OH P O

OH P OH

Protein (apoenzim)

Enzim (holoenzim)
Bukan Protein (gugus prostetik)

Koenzim (organik)

Kofaktor (Anorganik)

Ex: NADH, FADH, koenzim A, dan vitamin B.

Ex: Fe2+, Cu2+, Zn2+

Cofactors
Cofactors may be one of three types 1. Coenzyme: A non protein organic substance that is loosely attached to the enzyme 2. Prosthetic Group: A non protein organic substance that is firmly attached to the enzyme 3. Metal ion activators: K+, Fe2+, Fe3+, Cu2+, Co2+, Zn2+, Mn2+, Mg2+, Ca2+, or Mo2+,
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Types of Cofactors

Enzymes have varying degrees of specificity. One cofactor may serve many different enzymes.
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Enzyme Specificity

The action of an enzyme depends primarily on the tertiary and quaternary structure of the protein that constitutes the enzyme. The part of the enzyme structure that acts on the substrate is called the active site. The active site is a pocket in the enzyme structure where the substrate can bind.

Active Site

A restricted region of an enzyme molecule which binds to the substrate.

Substrate

Enzyme

Active Site
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19

How enzymes act as catalyst

General

Principles: The Induced Fit Model The lock-and-key model: proposed by Emil Fischer in
1894

The induced fit model: proposed by Daniel Koshland

in 1958, an enzyme induces a bound substrate molecule to adapt a conformation resembling the transition state

The Lock and Key Hypothesis

Fit between the substrate and the active site of the enzyme is exact Like a key fits into a lock very precisely The key is analogous to the enzyme and the substrate analogous to the lock. Temporary structure called the enzyme-substrate complex formed Products have a different shape from the substrate Once formed, they are released from the active site Leaving it free to become attached to another substrate

The Lock and Key Hypothesis

S E E Enzyme may be used again P P Reaction coordinate E

Enzymesubstrate complex

The Lock and Key Hypothesis

This explains enzyme specificity This explains the loss of activity when enzymes denature

SPECIFIC
COOH C H C H COOH

+H N 3

C CH2

NH4+
ASPARTASE

COOH FUMARATE COOH

COOH L-ASPARTATE COOH H ASPARTASE

C C

C CH2

NH3+

H ASPARTASE

COOH MALEATE

COOH D-ASPARTATE

The Induced Fit Hypothesis

Some proteins can change their shape (conformation) When a substrate combines with an enzyme, it induces a change in the enzymes conformation The active site is then moulded into a precise conformation Making the chemical environment suitable for the reaction The bonds of the substrate are stretched to make the reaction easier (lowers activation energy)

The Induced Fit Hypothesis

Hexokinase (a) without (b) with glucose substrate

http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html

This explains the enzymes that can react with a range of substrates of similar types

Enzymes and Reaction Rates

Factors that influence reaction rates of Enzyme catalyzed reactions include 1. Enzyme and substrate concentrations 2. Temperature 3. pH

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SUBSTRATE CONCENTRATION

At low concentrations, an increase in substrate concentration increases the rate because there are many active sites available to be occupied
At high substrate concentrations the reaction rate levels off because most of the active sites are occupied
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Substrate concentration

The maximum velocity of a reaction is reached when the active sites are almost continuously filled. Increased substrate concentration after this point will not increase the rate. Vmax is the maximum reaction rate
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Substrate concentration

Vmax is the maximum reaction rate The MichaelisMenten constant,Km is the substrate concentration when the rate is Vmax Km for a particular enzyme with a particular substrate is always the same
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Enzyme Kinetics
Km reflects the affinity of the enzyme for the substrate.
a) small km: reflects a high affinity of the enzyme for the substrate b) large km: reflects a low affinity of the enzyme for the substrate

Persamaan Michaelis-Menten

Vmax [S] V= ---------Km + [S]

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Michaelis-Menten Kinetics
if [S] << Km , vo = (Vmax/Km)[S]

if [S] = Km
if [S] >> Km

,
,

vo = 0.5 Vmax
vo = Vmax

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Example 1
The rate of an enzyme catalyzed reaction is 35 mol/min at [S] = 10-4 M, (KM = 2 x 10-5). Calculate the velocity at [S] = 2 x 10-6 M. Work the problem (alias dikerjakan donk)

Example 2
Ten micrograms of carbonic anhydrase (MW = 30000) in the presence of excess substrate exhibits a reaction rate of 6.82 x 103 mol/min. At [S] = 0.012 M the rate is 3.41 x 103 mol/min.
a. What is Vmax ? b. What is KM ? Work these..

Enzyme Concentration (HOW ?????)

Rate of Reaction

Enzyme Concentration

Effect of Temperature

Higher temperature increases the number of effective collisions and therefore increases the rate of a reaction. Above a certain temperature, the rate begins to decline because the enzyme protein begins to denature
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5- 40oC Increase in Activity

Temperature
40oC - denatures

Rate of Reaction 0

10

20

30

40

50

60

<5oC - inactive

Effect of heat on enzyme activty

If you heat the protein above its optimal temperature bonds break meaning the protein loses it secondary and tertiary structure

Effect of heat on enzyme activty

Denaturing the protein

Effect of heat on enzyme activity

Denaturing the protein

ACTIVE SITE CHANGES SHAPE SO SUBSTRATE NO LONGER FITS Even if temperature lowered enzyme

cant regain its correct shape

The effect of temperature

For most enzymes the optimum temperature is about 30C Many are a lot lower, cold water fish will die at 30C because their enzymes denature A few bacteria have enzymes that can withstand very high temperatures up to 100C Most enzymes however are fully denatured at 70C

Effect of pH

Pepsin is most efficient at pH 2.5-3 while Trypsin is efficient at a much higher pH

Each enzyme has an optimal pH at which it is most efficient A change in pH can alter the ionization of the R groups of the amino acids. When the charges on the amino acids change, hydrogen bonding within the protein molecule change and the molecule changes shape. The new shape may not be effective.
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The effect of pH

Extreme pH levels will produce denaturation The structure of the enzyme is changed The active site is distorted and the substrate molecules will no longer fit in it At pH values slightly different from the enzymes optimum value, small changes in the charges of the enzyme and its substrate molecules will occur This change in ionisation will affect the binding of the substrate with the active site.

Inhibitors

Inhibitors are chemicals that reduce the rate of enzymic reactions. The are usually specific and they work at low concentrations. They block the enzyme but they do not usually destroy it. Many drugs and poisons are inhibitors of enzymes in the nervous system.

The effect of enzyme inhibition

Irreversible inhibitors: Combine with the functional groups of the amino acids in the active site, irreversibly. Examples: nerve gases and pesticides, containing organophosphorus, combine with serine residues in the enzyme acetylcholine esterase.

The effect of enzyme inhibition

Reversible inhibitors: These can be washed out of the solution of enzyme by dialysis. There are three categories : Kompetitif Non kompetitif Un kompetitif

Competitive Inhibitors

Competitive inhibition occurs when the substrate and a substance resembling the substrate are both added to the enzyme. The inhibitor blocks the active site on the enzyme stopping its catalytic action
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Competitive Inhibition
Cartoon Guide
Substrate

Direct Plots

Vmax

vo I
Vo/2

E
Compete for Inhibitor active site

Km Km

[S], mM

Equation and Description

Double Reciprocal

E + S ES E + P + I EI
[I] binds to free [E] only, and competes with [S]; increasing [S] overcomes Inhibition by [I].

Vmax unchanged Km increased 1/vo

Intersect at Y axis

1/ Vmax 1/[S]

1/Km

Non-competitive Inhibitors

Non-competitive inhibitors deactivate the active site of the enzyme. They alter the enzyme so that it can no longer bind to the substrate
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Non-competitive Inhibition
Cartoon Guide
S S

Direct Plots

E
I

Vmax

vo I

Vmax

Different site

Equation and Description

Double Reciprocal

E + S ES E + P + + I I EI + S EIS
[I] binds to free [E] or [ES] complex; Increasing [S] can not overcome [I] inhibition.

Km = Km

[S], mM

Vmax decreased Km unchanged 1/vo

I

Intersect at X axis

1/ Vmax 1/[S]

1/Km

Uncompetitive Inhibition
Cartoon Guide Direct Plots
S

E
I

Vmax

Vmax

Km Km

[S], mM

Equation and Description

Double Reciprocal

E + S ES E + P + I EIS
[I] binds to [ES] complex only, increasing [S] favors the inhibition by [I].

Both Vmax & Km decreased 1/vo

Two parallel lines

1/ Vmax 1/Km 1/[S]

Enzyme Inhibition (Summary)

Type of reaction Effect of inhibitor

None Competitive Uncompetitive

None Increases Km Decreases Km and Vmax

Noncompetitive (mixed) Decreases Vmax (may increase or decrease Km)