Transcript Enzymology Part 2

Enzymology
Part 2
PRINCIPLES OF ENZYMOLOGY
TRANSITION STATE THEORY:
Colliding molecules of the reactants must have sufficient energy to overcome
a potential energy barrier (the activation energy) to react
Factors affecting rate of a rxn:
a. Sustrate concentration
b. Temperature
c. pH
d. Inihibitors, effectors
A. SUBSTRATE CONCENTRATION
A
B
PART A ([S] is low while [E] is high)
•
•
•
Directly proportional
Initial velocity (0) increases as [S] is small
compared to [E])
If [E] is higher than [S] in this section of the
graph, the frequency of collision is high
PART B ([S] is high while [E] is low
•
•
•
•
Rate no longer depends on [S]
[S] higher than [E]
Active site is saturated with substrate
Frequency of collision no longer the
determining factor
Determination of 0
All the test tubes have the same contents except that each has a different
substrate concentration
Either monitor the disappearance of substrate or the formation of product.
Therefore can obtain the rate of disappearance of S or formation of P at
each substrate concentration

0

Vmax = maximum velocity (achieved when all the substrate molecules complex with E or
enzymes are saturated with substrates)
•
Km & Vmax are constants which are unique for a pair of enzyme and its substrate

Km
= initial velocity
= Michaelis constant (takes into account all the rxn constants of the rxns involved
k1
E+S
k3
ES
k2
•
E+P
Km = (k2+k3)/k1
k4
Km = substrate concentration at ½Vmax
•
Units of Km = dm-3
•
Vmax [S]
o =
-------------Km + [S]
MICHAELIS MENTEN EQUATION
• Km can be determined graphically
• Km shows the affinity of an enzyme
for a substrate
• Km & Ks inversely related
• The bigger the value of Km, the lower the affinity
• Small Km value, the higher the affinity
LINEWEAVER-BURK PLOT
Km and Vmax can be determined from the  vs [S]
The Michaelis Menten equation can be modified to obtain a more Km and
Vmax precise values
1/0 = [Km/Vmax]. 1/[S] + 1/Vmax
ENZYME ACTION CAN BE CONTROLLED BY:
Non covalent inhibition:
2. Competitive inhibition
3. Non competitive inhibition
4. Uncompetitive inhibit
5. Covalent inhibition: Irreversible
6. Allosteric Control
COMPETITIVE INHIBITION
 Inhibitor competes with the substrate for the active site of the same
enzyme

Inhibitor and substrate have similar chemical structures

Lack of specificity at the active site: Cannot differentiate inhibitor from
substrate

Inhibitory effect can be overcome by increasing substrate concentration
KINETICS OF COMPETITIVE INHIBITION
Note that:
Vmax (- I) denoted V’max (in
the presence of Competitve
I)
Km < K’m
Because I and S compete for
the same site. More S is
required to achieve half
saturation point. Hence Km
increases.
K’m is more than Km by a
factor of (I + [I]/Ki)
V’maks = Vmaks (I + [I]/Ki)
When enzyme is saturated
with I, the effect can be
overcome by increasing [S].
So when the enzyme
becomes saturated with the S
then Vmax is achieved.
Example: inhibition of folic acid synthesis by sulphanamide
PABA (para amino benzoic acid
Sulphanamide
 PABA is required for the synthesis of folic acid
 Sulphanamida is a drug that competes with PABA for the enzymes in the folic acid
synthesis pathway.
Find another example of competitive inhibition in the cell
Non-Competitive Inhibition
1. The inhibitory effects cannot be overcome by increase in Substrate
concentration
2. Inhibitor binds to a site other than the active site. Binding of I does not affect
binding of S
3. Therefore in this case, structure of inhibitor is not similar to substrate
4. Inhibitory effects depend on I and Ki and not on [S]
5. I can bind to either E or ES
I
EI
 Gradient = Km/Vmax
 I/V’max = (I + [I]/KI)(I/Vmax
 Vmax reduces in the presence of
 Km does not change
non competitive inhibitor. It is as
though the amount of E is now less
Uncompetitive Inhibition
1. Binds only to the ES complex but not the free enzyme
2. Increasing [S] will increase the [ES] thus increasing [S] will not reverse the
effects of an uncompetitive inhibitor.
3. Lineweaver Burke plots will give a set of parallel lines
Note:
Both intercepts change but slope remains constant
Irreversible Inhibition
1. Enzymes can be inhibited by an irreversible manner for example by covalent
attachment either to E or ES
2. Kinetic pattern looks like non-competitive inhibition (net effect is a loss of
active enzyme): Vmax decreases
3. Reaction is time dependent decrease in enzymatic activity ie not
instantaneous as seen in non competitive inhibition
4. Penicillin is an irreversible inhibitor: binds to serine residue in the active site
of a the enzyme (glycoprotein peptidase). Affects cell wall synthesis, making
bacterial cells susceptible to rupture.
5. Others include: Hg2+, Pb2+, arsenic
6. Binds to functional groups such as: –COOH, -NH2, -SH dan -OH
Effect of Temperature:

Temperature increases the rate of reaction

Reaction rates increase because of the increase in collision/min between
the substrates

BUT at extreme temperatures, enzyme activity decreases because of
enzyme denaturation
Effect of pH
•
Influences ionisation of functional groups in proteins
•
As pH changes, the ionisation state of the functional groups of
the amino acids in the tertiary structure and in the active site will
change affecting enzyme activity
ENZYME REGULATION
Enzyme activity can be regulated by:
1. Covalent modificatio: eg phosphorylation of protein kinase
2. Zymogens
3. Allosteric regulation: non covalent interaction between enzymes
and small molecules (metabolites)
In allosteric regulation enzyme activities controlled at key steps in metabolic
pathways:
Feedback inhibition (feedback regulation)
The enzyme involved is a regulatory enzyme
Characteristics of Allosteric Enzymes
1. They have 2 binding sites: the active site and the modulator site
2. High molecular weight
3. Very complex
4. Difficult to purify
5. Contains 2 or more polypeptide chains (subunits): more than 1 S- binding
site/enzyme molecule
6. Does not obey Michaelis Menten kinetics:  vs [S] yields a sigmoid graph
compared to a hyperbolic curve
The binding of 1 substrate
to a protein molecule
makes it easier for
additional substrate
molecules to bind to the
same protein: substrate
binding is termed
cooperative
Characteristics of Allosteric Enzymes (con’t…)
6.
Allosteric enzymes are regulated by activation: ie there are effectors or modulators
that can have a positive (stimulatory) or negative effects on enzyme activity
POSITIVE COOPERATIVE EFFECT
and
NEGATIVE COOPERATIVE EFFECT
These enzymes take some time to reach
saturation point.
MODEL TO EXPLAIN ALLOSTERIC ENZYME
KINETICS
1. SYMMETRY MODEL: Monod, Wyman and
Changeux
a. Allosteric proteins exist in two conformational
stages:
R = Relaxed (high affinity for substrate) and
T = Taut (low affinity for substrate
b. Model named symmetry because in each
protein molecule all the subunits have either R
or T conformation
c. These 2 states are in equilibrium
R0
T0
1. SYMMETRY MODEL: Monod, Wyman aand Changeux (con’t…)
d. Presence of S will result in binding to R0 to form R1. This reduces the R0
concentration of disturbing theT0/R0 equilibrium. To restore equilibrium,
molecules in the T0 conformation will change to the conformation R0
e. Positive homotropic effectors
f.
This model also provides for binding to positive and negative effectors. If
effctors are not substrates then they are known as heterotropic effectors
g. Effectors that promote Substrate binding are known as positive
heterotropic effectors
h. Effectors that diminish Substrate
binding are known as negative
heterotropic effectors
i. Positive effectors increase number
of available binding sites
j. Negative effectors decrease number
of available binding sites
2. SEQUENTIAL MODEL: Koshland, Nemethy and Filmer
(Involves cooperativity and Conformational Changes)
Basis:
1. Protein molecules are not in symmetry (ie asymmetric).
2. Proteins are flexible molecules and conformations altered when ligands
bind
3. Binding of a ligand to one subunit of a multimeric protein, would cause
conformational changes to occur, and then through contacts with the other
subunits cause their conformation to change.
4. As a consequence other subunits will have either greater or lesser affinity
for the ligand
Aspartate Transcarbomylase