Enzim:

Mekanisme dan Katalisis

 Enzymes DO NOT change the equilibrium constant of a reaction
Enzymes DO NOT alter the amount of energy consumed or liberated in
the reaction (standard free energy change, G)
Enzymes DO increase the rate of reactions that are otherwise possible
Enzymes DO decrease the activation energy of a reaction (G)
 Enzymes DO increase the rate of reactions that are otherwise possible
Enzymes DO decrease the activation energy of a reaction (G)

The classic way that an enzyme

increases the rate of a bimolecular
reaction is to use binding energy to
simply bring the two reactants in close
proximity. In order for a reaction to
take place between two molecules, the
molecules must first find each other.
This is why the rate of a reaction is
dependent upon the concentrations of
the reactants, since there is a higher
probability that two molecules will
collide at high concentrations. The
enzyme organizes the reaction at the
active site, thereby reducing the cost in
terms of ENTROPY.
 How do enzymes catalyze biochemical reactions?
involves basic principles of organic chemistry

What functional groups can be involved in catalysis?

almost all alpha amino and carboxyl groups are tied up in peptide
bonds
R groups are involved in catalysis
asp, glu
his, lys
ser, cys, tyr

catalysis occurs when substrate is immobilized near these residues at

the active site
General Acid-Base Catalysis

General acid-base catalysis is

involved in a majority of
enzymatic reactions. General
acidbase catalysis needs to be
distinguished from specific
acidbase catalysis.

In General acidbase catalysis,

the buffer aids in stabilizing the
transition state via donation or
removal of a proton. Therefore,
the rate of the reaction is
dependent on the buffer
concentration, as well as the
appropriate protonation state.
General Base Catalysis I:
Ester Hydrolysis
 General Base Catalysis II:
Ester Hydrolysis

The hydrolysis of esters proceeds readily under in the presence of

hydroxide. It is base catalyzed. However, the rate of hydrolysis is
also dependent on imidazole buffer concentration. Imidazole can
accept a proton from water in the transiton state in order to generate
the better nucleophile, hydroxide. It can also re-donate the proton
to the paranitrophenylacetate in order to generate a good leaving
group.
 General Acid Catalysis :
Ester Hydrolysis

Electrostatic interactions are much

stronger in organic solvents than in
water due to the dielectric constant of
the medium. The interior of enzymes
have dielectric constants that are similar
to hexane or chloroform
 Catalysis by Metal Ions Catalysis I:
Ester Hydrolysis

Metal ions that are bound to the

protein (prosthetic groups or
cofactors) can also aid in
catalysis. In this case, Zinc is
acting as a Lewis acid. It
coordinates to the non-bonding
electrons of the carbonyl,
inducing charge separation, and
making the carbon more
electrophilic, or more susceptible
to nucleophilic attack.
 Catalysis by Metal Ions Catalysis
II:
Ester Hydrolysis

Metal ions can also function to make

potential nucleophiles (such as water)
more nucleophilic. For example, the
pka of water drops from 15.7 to 6-7
when it is coordinated to Zinc or
Cobalt. The hydroxide ion is 4 orders
of magnitude more nucleophilic than is
water.
 Covalent Catalysis :
Acetoacetate Decarboxylase

H 3C + O C O
H 3C CH 3
N H3 O
Lys
H 2C
O hydrolysis
O
CH 3
Lys N
H
CH 3

B
H 2O

CH 3
Lys N
H
CH 2
CH 3
O
Lys N
O H
CH 2

CO2 BH
 Enzymes physically interact with their substrates to effect
catalysis

E + S ES ES* EP E + P
where
E = enzyme
S = substrate
ES = enzyme/substrate complex
ES* = enzyme/transition state complex
P = product
EP = enzyme/product complex
Enzyme and substrate combine to form a complex

Complex goes through a transition state (ES*)

bound substance is neither substrate nor product

A complex of the enzyme and the product is formed

The enzyme and product separate

All of these steps are governed by equilibria

 Substrates bind to the enzymes active site
pocket in the enzyme

Substrates bind in active site by

hydrogen bonding
hydrophobic interactions
ionic interactions
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 Enzyme/Substrate Interactions
Lock and key model Induced fit model
substrate (key) fits into a substrate fits into a space in the
perfectly shaped space in the enzyme, causing the enzyme to
enzyme (lock) change conformation
change in protein conformation
leads to an exact fit of substrate
with enzyme

Following catalysis, the product(s) no longer fits the active site and is
released
 Strain and Distortion model
The binding of the
substrate results in the
distortion of the substrate in
a way that makes the
chemical reaction easier.
 Enzyme Kinetics

The rate of the reaction catalyzed by enzyme E

A+BP

is defined as
-D[A] or -D[B] or D[P]
Dt Dt Dt

Enzyme activity can be assayed in many ways

disappearance of substrate
appearance of product
continuous assay
end point assay

For example, you could measure

appearance of colored product made from an uncolored substrate
appearance of a UV absorbent product made from a non-UV-absorbent
substrate
appearance of radioactive product made from radioactive substrate
 Higher temperature generally causes more
collisions among the molecules and therefore
increases the rate of a reaction. More collisions
increase the likelihood that substrate will collide
with the active site of the enzyme, thus
increasing the rate of an enzyme-catalyzed
reaction.

Each enzyme has an optimal pH. 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.

The diagram shows that pepsin functions best in an acid

environment. This makes sense because pepsin is an
enzyme that is normally found in the stomach where the
pH is low due to the presence of hydrochloric acid.
Trypsin is found in the duodenum, and therefore, its
optimum pH is in the neutral range to match the pH of
the duodenum.
 At lower concentrations, the active sites on most of the
enzyme molecules are not filled because there is not
much substrate. Higher concentrations cause more
collisions between the molecules. With more
molecules and collisions, enzymes are more likely to
encounter molecules of reactant.
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. Reaction rate therefore increases
as substrate concentration is increased but it levels off.

If there is insufficient enzyme present, the

reaction will not proceed as fast as it otherwise
would because there is not enough enzyme for
all of the reactant molecules. As the amount of
enzyme is increased, the rate of reaction
increases. If there are more enzyme molecules
than are needed, adding additional enzyme will
not increase the rate. Reaction rate therefore
increases as enzyme concentration increases
but then it levels off.
 The velocity (V) of an enzyme-catalyzed reaction is dependent upon
the substrate concentration [S]

A plot of V vs [S] is often hyperbolic (Michaelis-Menten plot)

 The Michaelis-Menten equation describes the kinetic behavior of many
enzymes

This equation is based upon the following reaction:

SP

k1 k2
E + S ES E + P
k-1

V = Vmax [S]
KM + [S]

the reverse reaction (P S) is not considered because the equation

describes initial rates when [P] is near zero
 V = Vmax [S]
KM + [S]

V is the reaction rate (velocity) at a substrate concentration [S]

Vmax is the maximum rate that can be observed in the reaction

substrate is present in excess
enzyme can be saturated (zero order reaction)

KM is the Michaelis constant

a constant that is related to the affinity of the enzyme for the substrate
units are in terms of concentration

KM = k-1 + k2
k1
Enzyme (E) Substrate (S) K_M (in M) k_{cat} (in s^{-1}) k_{cat}/K_M (in M^{-1}s^{-1})
Acetylycholine esterase Acetylcholine 9.5 x 10^{-5} 1.4 x 10^4 1.5 x 10^6
Carbonic Anhydrase CO_2 0.012 1.0 x 10^6 8.3 x 10^7
Carbonic Anhydrase HCO_3- 0.026 4.0 x 10^5 1.5 x 10^7
Catalase H_2O_2 0.025 1.0 x 10^7 4.0 x 10^8
Fumerase Fumerate 5.0 x 10^{-6} 800 1.6 x 10^8
Fumerase Malate 2.5 x 10^{-5} 900 3.6 x 10^7
Urease Urea 0.025 1.0 x 10^4 4.0 x 10^5
 KM is also the substrate concentration at which the enzyme operates at
one half of its maximum velocity

if [S] = KM

V = Vmax [S]
2[S]

V = Vmax
2
 To determine KM and Vmax, decide on a number of different [S] values,
and measure V at each concentration (hold [E] constant)
 Michaelis-Menten plot is not useful for estimating KM and Vmax
it is better to transform the Michaelis-Menten equation to a linear form
actual values for KM and Vmax determined from graph
double reciprocal plot or a Lineweaver-Burk plot
 By taking the inverse of the Michaelis-Menten equation

V = Vmax [S]
KM + [S]

1 = KM + [S]
V Vmax [S] Vmax [S]

1 = KM . 1 + 1
V Vmax [S] Vmax

same form as y = mx + b
plot is y vs x
y is 1/V
x is 1/[S]
KM/Vmax is slope
y intercept is 1/Vmax
x intercept is -1/ KM
 Enzyme Inhibition

Certain compounds inhibit enzymes

decrease the rates of their catalysis
inhibition can be reversible or irreversible
3 types of reversible inhibitors
competitive inhibitors
non-competitive inhibitors
un-competitive inhibitors
Irreversible inhibition
suicide inhibitors
the various types of inhibitors can be
distinguished by the kinetics of their inhibition
 Competitive inhibition
inhibitor mimics substrate
fits into active site
malonate is a competitive inhibitor of
succinate dehydrogenase
 Competitive inhibitors can be identified by the kinetics of their inhibition
In the presence of a competitive inhibitor
KM increases
Vmax stays the same

The effects of competitive inhibition can be overcome by increasing [S]

 Non-competitive inhibition
inhibitor binds to a site other than the active site
Non-competitive inhibitors can be identified by
the kinetics of their inhibition
In the presence of a non-competitive inhibitor
KM stays the same
Vmax decreases
The effects of non-competitive inhibition
cannot be overcome by increasing [S]
 Un-competitive inhibition
inhibitor binds to a site other than the active
site, but only when substrate is bound
Un-competitive inhibitors can be identified by
the kinetics of their inhibition
In the presence of an un-competitive inhibitor
KM decreases
Vmax decreases
The effects of un-competitive inhibition cannot
be overcome by increasing [S]
 Irreversible inhibition
enzyme is covalently modified after interaction with inhibitor
derivatized enzyme is no longer a catalyst

Organofluorophosphates used as insecticides and nerve gases

irreversible inhibitors of acetylcholinesterase
form covalent product with active site serine residue
enzyme no longer functional
 When chymotrypsin is treated with DIPF
only ser 195 reacts is derivatized
other ser residues are not labeled
ser 195 is in the enzymes active site
Why is only ser 195 labeled?
adjacent amino acid residues in active site make ser 195 more reactive
 Coenzymes and Prosthetic Groups
some enzymes employ coenzymes and prosthetic groups at their active
sites
used for reactions that amino acid R groups cant perform
coenzymes
metals or small organic molecules
not covalently bound to protein
often function as co-substrates
precursors are often vitamins

prosthetic groups
small organic molecules
covalently linked to protein
 Enzyme Regulation :

1. Control of Enzyme Activity Level

A. Noncovalent modifiers cause conformational change between less active and more active states of
the enzyme.

B. Covalent Modification causes interconversion between inactive and active forms of the enzyme.

2. Control the Amount of the Enzyme

A. Isozymes - forms of the enzyme which differ in properties but catalyze the same reaction. For
example, enzyme forms which differ in Vmax and/or Km. The isozymes can be forms found in
different tissues and organs of an animal or for any eukaryotic organism, isozymes can be located in
different parts of the cell. For example, different isozymes of lactate dehydrogenase are found in
muscle and liver. Malate dehydrogenase occurs in different forms in the cytoplasm and the soluble
matrix phase of the mitochondria.

B. Biosynthesis of the enzyme protein can be controlled at the level of the gene via regulation of
transcription (ie synthesis of the enzyme's mRNA). This is more of a molecular biologic type of
regulation and involves molecules which bind to DNA and influence gene expression. This type of
control where the amount of the enzyme is governed can also be done after the mRNA is made, but
this is quite rare. In this mechanism, the mRNA is prevented from being translated and since mRNA is
rather unstable, it is degraded before it is effectively used by the ribosomes to make the protein.
 Allosteric Regulation

Control of Enzyme Activity by Non-Covalent Modifiers is usually called allosteric

regulation since the modifier binds to the enzyme at a site other than the active site
but alters the shape of the active site. Allosteric is a word derived from two Greek
words: 'allo' meaning other and 'steric' meaning place or site; so allosteric means
other site and an 'allosteric enzyme' is one with two binding sites - one for the
substrate and one for the allosteric modifier molecule, which is not changed by the
enzyme so it is not a substrate. The molecule binding at the allosteric site is not
called an inhibitor because it does not necessarily have to cause inhibition - so they
are called modifiers. A negative allosteric modifier will cause the enzyme to have
less activity, while a positive allosteric modifier will cause the enzyme to be more
active. In order for allosteric regulation to work, the enzyme must be multimeric (ie.
a dimer, trimer, tetramer etc.). The concept is easily illustrated using a dimer as the
model system, but it applies equally well to higher order multimers such as trimers
and tetramers, etc.
 Enzyme can bind two substrates
Cooperativity molecules at different binding sites.

k1 k2
S+E C1 P+E
k-1

k3 k4
S + C1 C2 P+E
k-3

or

S S

E C1 C2
S S
P P

E E
 The velocity (V) of an enzyme-catalyzed reaction is dependent upon
the substrate concentration [S]

For allosteric enzymes, a plot of V vs [S] shows a sigmoidal relationship

 Positive/negative cooperativity

Usually, the binding of the first

S changes the rate at which
the second S binds.
If the binding rate of the
second S is increased, its
called positive cooperativity
If the binding rate of the
second S is decreased, its
called negative cooperativity.
Lysozyme

Lysozyme is a small globular protein composed of 129 amino acids.

It is also an enzyme which hydrolyzes polysaccharide chains,
particularly those found in the peptidoglycan cell wall of bacteria. In
particular, it hydrolyzes the glycosidic bond between C-1 of N-acetyl
muramic acid and C-4 of N-acetyl glucosamine.
It is found in many body fluids, such as tears, and is one of the bodys
defenses against bacteria.
The best studied lysozymes are from hen egg whites and
bacteriophage T4.
Although crystal structures of other proteins had been determined
previously, lysozyme was the first enzyme to have its structure
determined.