Lecture 12 - Enzyme rate (Inhibition & Activation) Flashcards
The Michaelis-Menten equation
describes The V vs. [S] curve
V = Vmax [S] / Km + [S]
based on model reaction
model reaction
E + S ⟷ ES ⟶ E + P
⟷
- K1
- K-1
⟶
- K2
Vmax
how fast it can go if all enzyme is in ES complex
The more ES complex
faster it will go
Lineweaver - Burk Plot
X intercept
Y intercept
Double reciprocal
1 / S concentration vs 1 / V (kinetic rate)
Linear
-1 / Km
1 / Vmax
Significance of KM
substrate concentration needed to reach half Vmax
units mmol/ L
Characterises one enzyme-substrate pair (if an enzyme can act on different substrates, it will have different KM values for each).
For many enzymes k 2 «_space;k -1 , so approximation neglects k 2 :
Km = K-1 / K1
the ES dissociation constant
Low KM
high affinity between E and S;
high KM
low affinity between E and S;
Physiological significance of KM
in enzyme-substrate interaction,
[S] is below the KM.
rate will rise to accommodate more substrate, tending to maintain steady state.
KM: substrate preference and response
KM for each isozyme and substrate is same or different?
different
Isozyme glucokinase
stores energy as glycogen in the liver.
Turnover number, kcat
number of substrate molecules converted to product, per enzyme, per unit of time, when E is saturated with substrate.
define the activity of one enzyme molecule – a measure of catalytic activity.
If the Michaelis-Menten model fits , kcat = k2
kcat describes
the ‘rate limiting’ step.
Vmax = kcat [E]T
most effective enzymes should have…
A high kcat
A low KM
kcat / KM measure
enzyme efficiency;
the higher the better.
A high kcat
(ability to turnover a lot of substrate into product, per second).
A low KM
(low substrate concentration required to achieve near Vmax; high affinity for the substrate under the Michaelis-Menten assumptions).
kcat, KM and “catalytic perfection”
The upper limit for kcat / KM is the
diffusion-controlled limit; i.e. the rate at which enzyme and substrate diffuse together.
Viscosity of water sets an absolute upper limit at
~10^9 s-1 M-1.
Enzymes with kcat / KM above 10^8 s-1 M-1 are referred
to as
‘perfect’ catalysts.
Enzymes are optimized for
specific roles
Inhibitor:
compound that binds to an enzyme and reduces its activity.
why are Enzyme inhibitors Important?
o Natural inhibitors regulate metabolism.
o Many drugs, poisons & toxins are enzyme inhibitors.
o Used to study enzyme mechanisms.
o Used to study metabolic pathways.
Two classes of inhibitor
Irreversible inhibitor – binds covalently to the enzyme.
Reversible inhibitor – not covalently bound to the enzyme.
Irreversible inhibitor
binds covalently to the enzyme.
reacts with a specific amino acid side chain in the active site, and forms a covalent bond.
Reversible inhibitor
binds to the enzyme but can be released, leaving the enzyme in its original condition.
not covalently bound to the enzyme.
Competitive
or
Non-competitive (pure or mixed).
Covalent inhibitors often react with catalytic residues.
eg in chymotrypsin
Addition of the bulky tosyl-L-phenylalanine methylketone to the histidine disables the catalytic triad and fills the active site, blocking substrate binding.
Competitive inhibition definition
Inhibitor competes with the substrate for binding in
the active site.
Competitive inhibition
michaelis-menten plot
No change in Vmax:
high Km
High [S] than the
inhibitor.
Competitive inhibition
Lineweaver-burk plot
Increases KM:
Vmax unchange
More substrate is needed to get to V = Vmax / 2.
Transition state analogues as drugs
comp inhibitor
Enzymes are often targets for
drugs
Transition state analogues can make ideal
enzyme inhibitors.
Enalapril and Aliskiren lower
blood pressure.
Statins lower
serum cholesterol.
Protease inhibitors are
AIDS drugs.
Juvenile hormone esterase is a
pesticide target.
Tamiflu is an inhibitor of
influenza neuraminidase.
Transition state analogs make tight binding inhibitors
eg in Adenosine deaminase
use tetrahedral
intermediate.
• A non-reactive analog, 1,6-dihydroinosine, effectively inhibits the enzyme.
Substrate analogue inhibitors of HIV protease
•inhibitor fills the active
site.
• Two catalytic aspartic
acid residues.
Non-competitive inhibition
Inhibitor binds at a different site than the substrate.
Enzyme can bind substrate, or inhibitor (I), or both.
In pure non-competitive inhibition,
binding of I has no effect on the binding of S
i.e. the substrate binds to E and EI with the same affinity.
Competitive vs. pure non-competitive inhibition
michaelis menten plor
same Vmax / 2
different Km (comp more than pure)
Competitive vs. pure non-competitive inhibition
lineweaver burk plot
non
- km unchanged
- vmax reduce
comp
- km increase
- vmax unchange
Pure non-competitive inhibition
lineweaver burk plot
Vmax decreases;
KM stays the same.
Binding I changes the structure of the active site such that S still binds, but transition state stabilisation is no longer optimal.
Mixed non-competitive inhibition
Vmax decreases;
KM increases.
binding of the inhibitor does affect binding
of the substrate à mixed non-competitive inhibition.
Competitive inhibitors may react
alternate substrate can compete for the active site
of an enzyme,
as for alcohol dehydrogenase, on the right. Typically, related molecules fail to react., but some do.
Michaelis-Menten equation is based on
binding theory and simple chemical reaction rates.
Inhibition can be
reversible or irreversible.
Inhibition can change either
binding (KM) or
catalysis (kcat) or both.
Competitive inhibition gives a change in apparent
KM.
Competitive inhibitors often resemble
substrates or transition states.
