Enzim

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)

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.

Notice that without the enzyme it takes a lot more

energy for the reaction to occur. By lowering the
activation energy you speed up the reaction.

 Enzymes are proteins that act as biological

catalysts.
Cells use enzymes to speed up chemical
reactions that take place in cells.
Enzyme speed up reactions by lowering the
activation energies.
Because a particular enzyme catalyzes only
one reaction, there are thousands of different
enzymes in a cell catalyzing thousands of
different chemical reactions

Note:

Enzymes end in
ase
Based on the reaction they catalyze or the
substrate
For example:
Protease Protein
Amylase Starch (amylose)
Lipase Lipid

Classification
Group Assignment
Class is devided into 6 groups; each group is
responsible for one class of enzyme
Assigment should be submitted on the exam day.

 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 acid
base 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
O

H 3C

Lys

N H3

H 3C

CH 3

H 2C
O

hydrolysis

CH 3

Lys

N
H
CH 3
B

H 2O
CH 3

Lys

N
H
CH 2
CH 3

Lys

N
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

substrate (key) fits into a
perfectly shaped space in the
enzyme (lock)

Induced fit model

substrate fits into a space in the
enzyme, causing the enzyme to
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]
Dt

-D[B]
Dt

or

D[P]
Dt

Enzyme activity can be assayed in many ways

or

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

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

molecules at different binding sites.

Cooperativity
k1

S+E

k-1
k3

S + C1

k-3

C1

k2

C2

k4

P+E

P+E

or
S

C1

C2
S

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.