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Catalysis

Enzymes catalysts that allow biological reactions to


occur at a faster rate








Ea energy input required to initiate the reaction
Properties of A True Catalyst
Increases the rate of reaction by lowering the
activation energy barrier

Not used up or permanently changed during the
catalytic process

Does not change the position of equilibrium, only the
rate at which equilibrium is attained

Usually acts by forming a transient complex with the
reactant, thus stabilizing the transition state
Enzyme
Cofactor may be an organic or organometallic
molecule (coenzyme) or a metal ion ( Zn
2+
, Mg
2+
,

Cu
2+
)

Holoenzyme complete molecular package, protein
and cofactor

Apoenzyme protein component

Naming Enzyme
Common names for enzymes are usually formed by
adding the suffix ase to the name of the reactant
Tyrosinase catalyzes the oxidation of tyrosine
Cellulase catalyzes the hydrolysis of cellulose to produce
glucose

Some early enzyme names are less descriptive and
give no clue of their function or substrates
Trypsin, chymotrypsin
Enzyme nomenclature
6 major classes, each w/ subsclasses according to
rxn catalyzed

Each is assigned with a 4-digit number and
systematic name which identifies the rxn catalyzed.

Enzyme nomenclature
4-digit official number
1
st
digit: class name
2
nd
digit: subclass
3
rd
digit: accepting functional group
4
th
digit: accepting molecule

EC 1 Oxidoreductases
EC 1.1 Acting on CH-OH group of donors
EC 1.2 Acting on aldehyde or oxo group of
donors
EC 1.3 Acting on CH-CH group of donors
EC 1.4 Acting on CH-NH2 group of donors
EC 1.5 Acting on CH-NH group of donors
EC 1.6 Acting on NADH or NADPH

Kinetics
In a reaction of the form A + B P, the rate can be
expressed in terms of the rate of disappearance of
one of the reactants or in terms of the rate of
appearance of the product

Rate of disappearance of A = -A[A]/At where A symbolizes the
change on [A] and t is time

Rate of appearance of P = A[P]/At
So we can express rate in terms of any of these :
Rate = -A[A]/At = -A[B]/At = A[P]/At
Kinetics
It is established that Rate o [A]
f
[B]
g

Rate = k [A]
f
[B]
g

Where k is the rate constant (proportionality factor that accounts
for how well the reacting molecules oriented during collisions)

Exponents f and g usually small whole numbers 1 or 2 or in some
cases 0 must be determined experimentally
The overall order of the reaction is the sum of all the
exponents

Rate = k[A]
1
first order reaction w/ respect to A and first
order overall

Kinetics
First order reaction rate depends only on one
reactant

Second order reaction the rate of reaction depends
on the two reactants
Rate = k[A]
1
[B]
1
first order wrt A and first order wrt B and
second order overall

Zero order reaction rate is constant depends not on
concentrations of reactants but on other factors
(presence of catalyst)
A B, Rate = k[A]
0

Kinetic Properties of Enzymes
Initial rate (u
0
) determined during the first few
minutes of the reaction
Maximum velocity
(V
max
)

Michaelis-Menten Equation
Leonor Michaelis and Maud Menten explain the
hyperbolic rate curve
Proposed that enzyme molecules, E, and substrate molecules,
S, combine in a fast and reversible step to form an ES complex
E + S
k1
k2
ES
k3
k4
E + P where k are rate constants

Assumptions:
1. Neglect the reaction that reverts product P and free enzyme to
the ES complex defined by k4
2. ES complex is a steady-state intermediate
After mixing E and S, a certain level of ES is formed and its
concentration remain constant because it is produced at the
same rate as it breaks down

Time course of S
consumption, P
formation and
establishment of
steady state level of
ES

Bottom curve: detail
of early stage of time
course
M-M equation
] [
] [
max
0
S K
S V
M
+
= v
Km
Km Michaelis constant
In E + S
k1
k2
ES
k3
E + P, Km is expressed as:



Let Km = [S]

] [
] [ max
0
S K
S V
m +
= v
] [ ] [
] [
max
0
S S
S V
+
= v
] [ 2
] [
max
0
S
S V
= v
2
max
0
V
= v
1
3 2
k
k k
Km
+
=
Km
Remember:

Km becomes:

This is equal to the equilibrium constant for the dissociation of
the ES complex
ES
k2
k1
E + S



So when k2>>k3, Km is simply the dissociation
constant for the ES complex

1
3 2
k
k k
Km
+
=
1
2
k
k
Km =
1
2
] [
] ][ [
k
k
ES
S E
Keq = =
Km
Km measure of how tightly the substrate is bound
to the enzyme

The greater the value of Km, the higher is k2 than k1

Km is the inverse measure of the affinity of the
enzyme for the substrate

Vmax and K3
If the substrate concentration is so high so that E is
completely saturated : [ES] = [E
T
] and V= Vmax

From this part of the enzyme equation:
ES
k3
k4
E + P



Turnover number, k3 (kcat or kp) number of moles
of substrate transformed to product per mole of
enzyme in a defined time period
] [
max
3
T E
V
k =
] [ 3 ES k V =
] [ 3 max T E k V =
K
M
, K
cat
Carbonic anhydrase -CO
2
:
K
cat
= 1 x 10
6
s
-1
;
K
M
= 1.2 x 10
-2
M
Carbonic anhydrase -HCO
3
-
:
K
cat
= 4 x 10
5
s
-1
K
M
= 2.6 x 10
-2
M


Which is better substrate for the enzyme?
K
M
, K
cat
Carbonic anhydrase - CO
2
:
K
cat
= 1 x 10
6
s
-1
;
K
M
= 1.2 x 10
-2
M


Carbonic anhydrase - HCO
3
-

K
cat
= 4 x 10
5
s
-1
K
M
= 2.6 x 10
-2
M


K
cat
/K
M
= 8.3 X10
7
M
-1
s
-1
K
cat
/K
M
= 1.5 X10
7
M
-1
s
-1
Lineweaver-Burk Equation
Double reciprocal plot allows one to plot
experimental enzyme rate data in the form of a
straight line






y = m x + b
] [
] [ 1
max
0 S V
S K
M
+
=
v
max
max
0
1
] [
1 1
V S V
K
M
+ =
v
] [
] [
] [
1
max max
0 S V
S
S V
K
M
+ =
v
] [
] [
max
0
S K
S V
M
+
= v
max
max
0
1
] [
1 1
V S V
K
M
+ =
v
Other Methods of Km and Vmax
Determination
Eadie-Hofstee Plot
The Michaelis-Menten equation is rearranged to



This is a graph of u
0
vs u
0
/[S] where:
Slope = -Km
Y-intercept = Vmax
X-intercept = Vmax/Km
max
0
0
] [
V
S
Km + =
v
v
Eadie-Hofstee
Other Methods of Km and Vmax
Determination
Hanes-Woolf Plot
The Michaelis-Menten equation is rearranged to



This is a graph of [S]/u
0
vs [S] where:
Slope = 1/Vmax
Y-intercept = Km/Vmax
X-intercept = -Km
max max 0
] [
1 ] [
V
Km
S
V
S
+ =
v
Hanes-Woolf
Characteristics of Enzyme Reactions
1. Enzyme

2. pH enzymes have an optimal pH at which they
function most effectively (pH 6-8)
Characteristics of Enzyme Reactions
1. Enzyme

2. pH enzymes have an optimal pH at which they
function most effectively (pH 6-8)

3. Temperature enzymes are sensitive to
temperature changes

For most enzymes, the rate decline begins in the temp.
range of 50C to 60 C
Binding of Substrate to Enzyme
Active site specific region in the enzyme where the
substrate specifically binds

Pocket or crevice in the 3-D structure of the enzyme
Consists of certain amino acids that may be involved with the
noncovalent interaction with the substrate
Characteristics of The Active Site
1. Specificity it is able to discriminate among
possible substrate molecules
Two Types
1. Absolute accept only one type of molecule (can even
discriminate between a D or L isomer)

2. Group Specificity accept a number of closely related
substances as long as the reactive functional group is
present
Characteristics of The Active Site
2. Relatively small, 3-D region within the enzyme aa
residues need not be contiguous in the linear
protein chain

3. Holds substrate through weak, noncovalent,
reversible interactions hydorphobic, ionic and H-
bonding
First Step
Binding of substrate to the enzyme
Three Models
Lock and key model

Induced-fit model

Transition-state model
Lock-and-Key model
Induce-fit model
Transition state model
Increase in Reaction Rate








1. Entropy loss in the ES formation
2. Destabilization of ES due to strain, desolvation or
electrostatic effect
Loss of Entropy
AG = AH - TAS

ES complex is highly organized compared to E and S
in solution

E and S have translational entropy (freedom to
move in 3-D) as well as rotational entropy (freedom
to rotate or tumble about in an axis)

Destabilization of ES Complex
By strain or distortion consequence of the fact that
the enzyme is designed to bind the transition state
more strongly than the substrate

By desolvation of charged groups in the substrate
charged groups are highly stabilized in water

By electrostatic destabilization when a substrate
enters the active site, charged groups may be forced
to interact with groups with same charge resulting to
repulsion and destabilization
2
nd
Step
The transition state is formed and catalysis can occur

Proximity and orientation speed up the reaction in
the transition state, the substrate is bound close to
atoms with which it is to react and also placed in the
correct orientation wrt those atoms
Mechanistic Features of Enzymes
General Acid-Base
Catalysis
Step 1 H
+
is added to
the carbonyl group
Step 2 formation of
the tetrahedral
intermediate
Step 3 a proton is
transferred from O to
N
Step 4 a base will
assist by accepting the
proton from the
intermediate
Mechanistic Features of Enzymes
Metal-ion Catalysis alkali metal ions (Na+, K+)
and transition metals (Mg
2+,
Mn
2+,
Cu
2+
, Zn
2+
,

Fe
2+
,

Fe
3+
,

Ni
2+
,

and others)

1. Holds a substrate properly oriented by coordinate
covalent bonds
Mechanistic Features of Enzymes
Metal-ion Catalysis

2. Enhance reaction by polarizing the scissile bond or
by stabilizing a negatively charged intermediate
Mechanistic Features of Enzymes
Metal-ion Catalysis

3. Participate in biological oxidation-reduction
reactions by reversible electron transfer between
metal ions and substrate




Acts as a Lewis acid by accepting electrons
Mechanistic Features of Enzymes
Covalent Catalysis nucleophilic substitution
reaction
A nucleophilic, (electron-rich) functional group attacks an
electron-deficient group
Nucleophile Leaving
group
Active Site Events
Functional groups in the active site that can have
catalytic roles:

Imidazole ring of histidine
Hydorxyl group of serine
Carboxyl side chain of aspartate and glutamate
Sulfhydryl group of cysteine
Amine group of lysine
Phenol group of tyrosine

Mechanism of Chymotrypsin Action
Serine residue at position 195 is required for activity
Another critical aa in chymotrypsin is His 57
Ser 195
His 57
Asp 102
Enzyme Activity Regulation
Irreversible inhibitor forms covalent or very strong
noncovalent interactions with the enzyme
E-H + R-X E-R + HX
(active) (inactive)

Example: aspirin (acetylsalicylic acid)
Acts by blocking synthesis of pain-producing prostaglandins
Reversible Inhibitors
Can readily combine with and dissociate from an
enzyme and render the enzyme inactive only when
bound

EI is held by weak, noncovalent interactions similar
to ES complex
Competitive Inhibition
Inhibitor usually resembles the structure of normal
substrate and is capable of binding to the active site
of the enzyme





Transition state analogs modeled after the
structures of substrate in presumed transition states

Noncompetitive Inhibition
Inhibitor can bind to the enzyme at a site other than
the active site of the enzyme
Binding of inhibitor causes a change in the structure
of the enzyme especially around the active site
Uncompetitive Inhibition
Inhibitor binds only to the ES complex but not with
the free enzyme
Influence the activity of the enzyme only when [S] is
high and, in turn, the ES concentrations are high
Kinetics of Inhibition
Competitive inhibition slope and x-intercept of
Lineweaver-Burk plot change but the y-intercept
does not
Km increase more substrate is needed to get the velocity of
half the maximum velocity
Kinetics of Inhibition
Pure noncompetitive inhibition - slope and y-
intercept of Lineweaver-Burk plot change but the x-
intercept does not
Pure - Vmax decreases but Km remains the same because the
inhibitor does not interfere with the binding of substrate to the
active site

Mixed Vmax decreases and Km increase because the
inhibitor affects the binding of the substrate to the active site

Kinetics of Inhibition
Uncompetitive inhibition Vmax and Km both
decrease

Reversal of inhibition is not achieved by increase in
[S]
Allosteric Enzymes



Concentration of final products (feedback inhibition)
Concentration of the beginning substrate
Concentration of an intermediate formed in the
sequence
Concentration of external factors (hormones)

Effectors
Biomolecules that influence the action of an
allosteric enzyme
Positive effectors stimulants
Negative effectors - inhibitors
Catalytic and Regulatory Sites
Catalytic site where substrate binds

Regulatory site the binding of effector molecules
changes the conformation of the protein in a way
that tells the other subunits that it is bound
Chemical Alterations
1. Phosphorylation of hydroxyl groups of serine,
threonine or tyrosine

2. Attachment of an adenosyl monophosphate to a
hydroxyl group

3. Reduction of cysteine disulfide bonds
Catalyzed by phosphorylase kinase
Catalyzed by phosphorylase kinase
Catalyzed by phosphorylase phosphatase
Glutamine synthetase
Proteolytic Cleavage
Zymogen inactive protein precursor
Cleaved at one or a few specific peptide bonds to produce the
active form of the enzyme
Regulation by Isoenzyme
Isoenzymes multiple forms of enzymes that have
similar but not identical amino acid sequences

May demonstrate the same enzyme activity but the may differ
in kinetics (Km and Vmax), differ in effectors, differ in the
form of coenzyme needed and cellular distribution