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Donna D.

Smith
Biology Department
Northeastern State University
FREE ENERGY OF ACTIVATION

■ For a reaction A--->P, at any point in time a


portion of the A molecules contain enough
energy to attain an activated state, the
transition state, in which the probability is
very high that the reaction will proceed.
■ The transition state is at the top of the
energy barrier separating the reactants and
products.
Catalysts make reactions more probable.

■ Catalysts act to lower the activation


barrier.
■ Catalysts lower the free energy of
activation of the reaction which is defined
as the amount of energy to bring one mole
of reactants at a given temperature to the
transition state at a given temperature to the
transition state.
Activation Energy

Transition point

Non-catalyzed reaction

Free Activation energy


Energy Catalyzed reaction
reactants
products

Progress of reaction
Enzyme Mechanisms

■ 1. Proximity and orientation of substrate


Enzymes can bind substrate molecules and
thereby increase the local concentration
many times, 105 as great as the overall
enzyme concentration. REMEMBER:
Substrates in vivo are present at
concentrations much lower than saturation.
2. Covalent catalysis
A highly reactive covalent intermediate, the ES
complex, is formed in this mechanism with a
lowered activation energy.
Most common mechanisms for accomplishing this
is for a nucleophilic group on the catalyst to attack
an electrophilic group on the substrate. A
nucleophilic group contains electron rich atoms
which can donate electrons. On enzymes these are
serine, cysteine, and histidine.
Hydrolysis reactions are
frequently this type of reaction
Slow, non-catalyzed
ƒ RX + H2O -------------> ROH + X- + H+

Fast, catalyzed—2 steps:


ƒ 1. RX + H2O -------------> RY + X-
RY is a highly reactive intermediate
ƒ 2. RY + H2O --------------> ROH + H+ + Y
3. Acid base catalysis
The enzyme can act as a donor or
acceptor of protons. Proton donors at
physiological pH’s include any protonated
ionizable R-group, but histidine is most
likely to donate a proton. Proton acceptors
include any ionized R-group.
The pK of histidine makes it a potential
proton donor or acceptor at physiological
pH’s.
ƒ This property makes it an amino acid which
frequently is in the catalytic site of the enzyme.

ƒ Histidine also is an important nucleophile.


Histidines are conserved evolutionarily as are the
catalytic site sequences. Actually there are few in
most enzymes, but most frequently they are
located in the catalytic site.
4. Enzyme conformation
■ The analysis of the three dimensional structures of
enzymes has shown critical (essential for catalysis)
residues to be located near one another. This confirms the
notion of an actual physical catalytic site.

■ In addition, the conformational changes which occur when


the enzyme binds to the substrate cause the ES complex to
be converted to the transition state more readily by
distorting both the enzyme an substrate.
The binding of the substrate forces the enzyme into a
position more favorable for the reaction.
WHICH OF THESE 4 FACTORS
IS THE MOST IMPORTANT ?

PROXIMITY AND
ORIENTATION OF THE
SUBSTRATE
Chymotrypsin
A serine protease
Chymotrypsin is a digestive enzyme
synthesized in the pancreas.
ƒ It is a 25 kd protein composed of three
polypeptides joined by two interchain disulfide
bridges.
ƒ Chymotrypsin folds into a compact structure with
all charged amino acids on the outside except for
the residues which participate in the active site.
ƒ Chymotrypsin contains several regions of β
conformation, but little α helix.
Rasmol structure
Active site of chymotrypsin
Chymotrypsin binds its substrate with a
hydrophobic active site that recognizes large,
hydrophobic amino acid residues on the carboxyl
side of a peptide bond.
susceptible peptide bond
H
+H N C C N C C O 2-
3
amino terminus R O R' carboxyl terminus

hydrophobic R group
( phe, met, tyr, or trp )
Chymotrypsin belongs to a family of
proteins called the serine proteases.
■ All members of this family have similar
active sites, and thus similar reaction
mechanisms.
■ Trypsin and thrombin belong to this
important family.
Chymotrypsin catalyzes the hydrolysis
of peptide bonds in two distinct stages.
ƒ 1. ACYLATION. In this stage ser 195 of the
enzyme reacts with the carbonyl group of the
peptide bond being attacked to form an acyl-
enzyme intermediate. This releases the amine
component of the substrate.
ƒ 2. DEACYLATION. In this stage the acyl-
enzyme intermediate is hydrolyzed by water. This
releases the acid component of the substrate.

Chymotrypsin hand-out
Serine proteases: The catalytic triad

■ Three amino acids, ser195, his 57, and asp102, are correctly
positioned in three dimensions to allow ser 195 to launch a
nucleophilic attack on the peptide bond.
Enzyme alone
H
O C
Asp102 C O H N N H O Ser 195
C CH

His 57
with addition of substrate

O C
+
Asp102 C O H N N H O Ser 195
C CH
Substrate
His 57
Active site of chymotrypsin
Yellow substrate + CPK active site.

All CPK.
1st) Acylation: Formation of a covalent
intermediate with a lowered activation energy
H
O C
Asp 102 C O H N N H O Ser 195
C CH
R O
His 57
N C R'
H
H
O C tetrahedral transition state
Asp 102 C O H N N H O Ser 195
O
C CH C
R R'
His 57
N
H
H
O C acyl-enzyme intermediate
Asp 102 C O H N N O Ser 195
O
C CH C
R'
His 57 R
N H amine component of
H the substrate
2nd) Deacylation: Release of acid
component of the substrate
H
O C acyl-enzyme intermediate
Asp102 C O H N N O Ser 195
O
C CH H C
O
R'
His 57 H

H
O C tetrahedral transition state
+
Asp102 C O H N NH O Ser 195
HO
C CH C
O
R'
His 57

H
O C
Asp102 C O H N N HO Ser 195
C CH O
His 57 R' C O
acid component of
the substrate
Lysozyme
A glycosidase
Lysozyme cleaves the polysaccarides
of bacterial cell walls.
■ Lysozyme is a 14.6 kd polypeptide
stabilized by 4 disulfide bridges.
■ The secondary structure of lysozyme has
both β conformation and α helix.
■ It is compactly folded with only non-polar
residues on the interior.

Rasmol structure
Active site of lysozyme

■ Lysozyme binds 3 disaccarides at once.


■ It hydrolyzes the glycosidic bond
between the C-1 of NAM (4th
monosaccaride) and C-4 of NAG (5th
monosaccaride).
-NAG-NAM-NAG-NAM-NAG-NAM-
1 2 3 4 5 6
Susceptible glycosidic bond

susceptible β glycosidic bond

CH 2OH CH 2OH

NAM
O O
NAG
OH
O O
HO 1 4
OH
H 3C CH NH
NH
COO O C
O C
CH 3
CH 3
Active site of lysozyme
Enzyme + yellow substrate.

Yellow substrate + active site.

All CPK.
Mechanism of Action

1st) Formation of carbonium ion


2nd) Reaction with water
Formation of carbonium ion, a
stabilized intermediate

O
O 5
5
H C4
C4 O
O H Glu35 C O
Glu35 C O O
O C 1+
C1 O O
O O
4 C Asp 52
4 C Asp 52
O
O

Carbonium ion intermediate


Reaction with water

H+
O -
C OH OH
Glu35 Glu35 C OH
O O
C+ 1 C 1+
O O O O
4 C Asp 52 4 C Asp 52
O O
EFFECT OF pH ON ENZYME ACTIVITY

■ Most enzymes are active at a specific pH,


above and below this pH the activity
diminishes.

The pH optimum can be an important


means of controlling enzyme activity.
This is a typical activity curve for
trypsin.

Relative
activity

2 8
pH
EFFECT OF TEMPERATURE ON
ENZYME ACTIVITY

The rate of enzyme catalyzed reactions increases


with increases in temperature. For each 10oC rise in
temperature the rate of the enzyme’s activity
doubles.

Most enzymes are inactivated at temperatures above


55 to 60 oC.
Temperature vs. activity

100

% activity

50

20 40 60
Temperature oC