Enzyme Kinetics Flashcards
Speed-up chemical reactions in cells by…
placing the substrate in an environment
that facilitates the reaction
Enzymes are proteins with…
catalytic activity
– A catalyst increases the rate of a chemical reaction without being consumed
– 103
-1020 x faster than uncatalyzed reactions
– Exhibit stereospecificity
– Function under physiological conditions
What is Enzyme Kinetics?
- Study of the rates, mechanisms and
properties of enzyme-catalyzed
reactions - Provides information on enzyme
specificities, regulation and adaptation - Clinically important for diagnosis based on changes in enzyme activity or
amount
Enzymes are very powerful
Catalysis
Enzymes can distinguish between…
enantiomers
Some enzymes require
additional…
chemicals or
groups to function:
– Metal ions
– Prosthetic groups
* Heme
– Co-factors
* ATP
6 types of chemical reactions are catalyzed by enzymes:
– 1. Oxido-reduction: oxidoreductases
– 2. Transfer of chemical groups: transferases
– 3. Hydrolysis: hydrolases
– 4. Removal of chemical groups: lyases
– 5. Isomerisation: isomerases
– 6. Linking two groups together: ligases
Oxidoreductases:
Example: Lactate dehydrogenase
(EC 1.1.1.27)
- Lactate = substrate
- Pyruvate = product
- NAD+
/NADH = co-factor
- Transferases:
Example: Alanine transaminase
(EC 2.6.1.2)
- L-alanine / a-ketoglutarate = substrates
- Pyruvate / L-glutamate = products
- Hydrolases:
Example: diphosphate phosphohydrolase(EC 3.6.1.1)
- Pyrophosphate / H2O = substrates
- Phosphate = product
- Lyases (aka synthases):
Example: Pyruvate decarboxylase(EC 4.1.1.1)
- Pyruvate = substrate
- Acetaldehyde and CO2 = products
- Isomerases:
Example: Alanine racemase
(EC 5.1.1.1)
- L-alanine = substrate
- D-alanine = product
- Ligases (aka synthetases):
Example: L-glutamine synthetase(EC 6.3.1.2)
- L-glutamate / NH4
+
= substrates - L-glutamine = product
- ATP = co-factor
Enzymatic reactions take place in…
multiple steps
involving reaction intermediates.
- While in theory these reactions are reversible, in practice, the low levels of the one of the reactants (S or P) usually pushes the equilibrium in one direction.
Enzymes increase the rate of …
chemical reactions, but do NOT alter the direction of the equilibrium.
In chemical reactions, three conditions
must be met for a reaction to take place:
- the molecules must collide to react
- there must be enough energy
(energy of activation) for the two
molecules to react. - the molecules must be orientated
correctly with respect to each other.
- the molecules must collide to react.
If two molecules simply collide, however,
they will not always react; therefore, the
occurrence of a collision is not enough.
- there must be enough energy
(energy of activation) for the two
molecules to react.
If two slow molecules
collide, they might bounce off one another
because they do not contain enough
energy to reach the energy of activation
and overcome the transition state (the
highest energy point).
The transition state is not only a reaction
intermediate:
it is a transitory molecular
structure that is no longer the substrate, but
not yet the product.
In the absence of an enzyme, the substrate requires
a
substantial amount of energy in order to reach the activation state and react
In the presence of an enzyme, the reaction is facilitated because…
the enzyme provides a better environment for the reaction to occur:
– Close proximity of substrate and chemical groups of the enzyme
– Proper orientation of the chemical groups with respect to the substrate
– The formation of the transition state is favoured
The « lock and key » thing is a…
a MYTH:
– If the enzyme and substrate were perfectly
complementary, like a lock and key, the interaction between E and S would be so stable that the
reaction would not occur!
- Instead, the 3D shape of the enzyme is complementary to the
transition state; - By doing so, the enzyme favours the formation of the transition
state, lowers the energy of activation, and accelerates the
reaction…COOL!
Factors influencing enzyme activity
- Temperature
- pH
- Substrate concentration
- Product concentration
- Presence of inhibitors (or activators)
- Temperature Factor:
Reflects weak interactions holding
native conformation together
- pH Factor
Due to presence of charged amino acids
in the active site
How do we measure enzyme activity?
- Detection of the product(s):
– pNA = para-nitroaniline Absorbs at 405 nm - Accumulation/utilisation of a co-factor:
– NADH = absorbs strongly at 340 nm (e = 6.3 mol.L-1cm-1)
– NAD+
=does not absorb at 340 nm - Coupled reactions:
– Very useful when neither substrate/product/co-factor can be (easily) detected directly
v0
(initial velocity) is the…
rate of the reaction very early on when [P] is negligible
v0
can be obtained by…
taking the slope of
the graph of [P] vs Time
(units: concentration / time)
v0
varies as a
function of [E]
v0
increases as a function of…
[S] until E is
saturated by S
When E is saturated with…
S –> v0 = Vmax
Michaelis-Menten Equation:
The relationship between vo
and [S] can be viewed as a 2
step reaction.
This relationship can be
expressed by the MichaelisMenten equation.
Michaelis-Menten Equation (Km):
Km can be calculated as the [S] required to acheive half the Vmax
- Km is a measure of the affinity of E for S
– The lower the Km, the less S is requried by E to acheive ½ Vmax, and the greater the affinity of E for S.
Turnover number is the…
maximum number of substrate
molecules converted to product per second by each active site (units = s-1)
The inverse is the amount of time required for E to convert 1 substrate
molecule to the product (i.e. time for 1 catalytic event). Units: s.
Neither Km nor Vmax can
be easily obtained …
directly from kinetic
data because Vmax is
rarely acheived (its an
hyperbolic curve…)
Michaelis-Menten equation describes the relationship between…
reaction rate and substrate concentration. It can explain the
saturation behavior in catalyzed reactions as shown in the
previous slide.
Michaelis-Menten equation is derived based on the following
three conditions:
1) State steady assumption;
2) Initial velocity assumption;
3) Rate law.
When enzyme is mixed with a high
concentration of substrate [S], the…
reaction
goes rapidly to steady state.
– Does not allow characterization
Vmax is asymptotically approached as…
substrate is increased, but
never reached in reality, because this requires that ALL enzymes are bound with substrate (think about chemical equilibrium);
Two main types of inhibition exist:
– Reversible enzyme inhibition: enzyme activity can be
recovered by removing the inhibitor (e.g. dialysis, gel filtration)
– Irreversible enzyme inhibition: inhibitor binds covalently to
enzyme, which is then irreversibly inactivated
VEGF (Vascular Endothelial Growth Factor):
Example of enzyme inhibition.
– Produced in embryos and tumours;
– Acts via a cell surface receptor to trigger the
growth of blood vessels;
– Why inhibit VEGF-R:
* Blocking the action of VEGF (an enzyme)
will block the growth of blood vessels and
starve tumours to death!
Sildenafil:
Example of enzyme inhibition
– cGMP-Phosphodiesterase
inhibitor;
– Initially tested as an
anti-hypertension drug
-
1- Competitive inhibition
- Most frequently encountered
inhibitors; - I is very similar to S
– (i.e. it is a structural analog) - I and S compete for the same
binding site on the enzyme: the
active site; - Vmax stays the same:
– At high enough [S], S will
outcompete I - Km is increased (Km
app):
– Because I can bind E, the
amount of S required to reach
½ Vmax will be increased.
2- Uncompetitive inhibition
- I only bind to ES, not the free enzyme;
- Example: glyphosate (Round-up herbicide)
- Vmax is decreased:
– Some of the E is converted into an
inactive ESI complex. - Km is decreased:
– I reduces the amount of E that can
participate in the reaction;
– ESI shifts the E + S ES to the right,
leading to an apparent decrease in Km.
3- Noncompetitive inhibition:
- I and S bind to different sites on E;
- Binding of I on E does not affect the
binding of S on E (and vice versa); - So: Km is unchanged, but Vmax is
decreased (I reduces the [E] that
can generate P); - e.g. deoxycyclin (an antibiotic), that
inhibits collagenase (a proteolytic
enzyme involved in periodontal
diseases).
irreversible inhibitors bind covalently to the…
enzyme and permanently inhibit it.
Irreversible enzyme inhibition types:
– Group-specific
– Active site-directed reagents (aka Affinity labels)
– Suicide inhibitors
Three main types of catalysis are found:
General acid/base
catalysis:
Metal ion catalysis:
Covalent catalysis:
General acid/base
catalysis:
Involves the transfer of
protons between
side chains of the
enzyme and the
substrate;
The most common form
of enzyme catalysis
Metal ion catalysis:
Some enzymes use metal
ions for catalysis (Mg+2
, Mn+2, Zn+2, Fe+2, Cu+2)
The metal ion can act is
different ways:
Stabilise transition state
Help orientate the substrate
vs enzyme
Participate in transfer of
electrons/protons
between enzyme and
substrate
Covalent catalysis:
Involves the formation of a transientcovalent bond between the enzyme and
substrate;
Covalent bond activates substrate for further reaction
In any organism, enzymes are always kept in check so that their activity can match the needs of the cell:
– Enzymes can be activated: the reaction is stimulated to generate more product;
– Enzymes can be inactivated (or inhibited): the reaction is slowed down to
decrease the amount of product;
- Several strategies are used to modulate (i.e. activate or inhibit) enzymes:
– 1. Allostery
* Inhibition by product
* Activation by substrate/cofactor
– 2. Binding of regulatory subunits
– 3. Covalent modification* Phosphorylation on Ser/Thr/Tyr
– 4. Degradation of the enzyme
– 5. Limited proteolysis–
- Allostery
binding of an effector molecule to a separate site on the enzyme
Allostery is widely used by
metabolic enzymes:
– Inhibition by the end product of a pathway;
– Activation by a product generated early on in the
pathway.
(Allostery) Based on the principle of cooperativity:
– The binding of a small molecule to the enzyme
modifies the 3-D structure of the protein and altersits ability to catalyse the reaction.
- Regulatory subunits
- cAMP is produced from ATP by the
action of adenylate cyclase. - The binding of cAMP to the
regulatory subunits of PKA frees
the catalytic subunits, that are
then fully active.
- Regulation by covalent modification:
- Specific amino acid side chains of several enzymes are the target of covalent modifications (catalysed by yet other
enzymes…).
Regulation by phosphorylation:
Addition of a
phosphate group
(phosphorylation) by
protein kinases
or
their removal by
protein phosphatases
is frequently used to
modulate the activity of
enzymes
- Regulation of enzyme stability:
- Proteins are constantly being made (synthesized) and
destroyed (degraded). - The tight regulation of protein synthesis and degradation is a key factor in the regulation of enzyme activity.
- Regulation of enzyme stability (Ubiquitin:)
– 76 amino acid protein
– Tags other proteins for degradation
- Ubiquitin is attached to other proteins by a
series of 3 enzymes (E1, E2 and E3)
- Regulation of enzyme stability: Polyubiquitylated proteins
are targeted to
a HUGE protein complex called the
proteasome:
– Contains several subunits with different
types of protease (i.e. protein hydrolases)
activities.
Degradation of an enzyme results in a
decrease in the…
amount of its product that
can be made.
- Limited proteolysis:
- Several enzymes (particularly digestive
enzymes) are initially synthesized as
inactive precursors (called zymogens /
proenzymes). - Activation of the enzymes is done by the
cleavage of a limited number of peptide
bonds (usually 2-3); - The mature enzyme is made up of 2 to 3
chains, held together by disulfide bonds
- Limited proteolysis: ( Pancreatic trypsin inhibitor)
- inhibits trypsin, and prevents trace
amounts of activated trypsin from triggering the proteolytic cascade in the pancreas/pancreatic ducts; - Inherited deficiencies in a similar protease inhibitor (a1-antitrypsin, which inhibits elastase), leads to damage to the lungs and
emphysema.
