Transcript Mechanism of enzyme action, kinetic of enzymatic catalysis

Mechanism of enzyme action, kinetic of
enzymatic catalysis.
Kinetic properties of enzymes
Study of the effect of substrate concentration on the rate of reaction
Leonor Michaelis and Maud Menten – first researchers
who explained the shape of the rate curve (1913)
During reaction enzyme molecules, E, and substrate
molecules, S, combine in a reversible step to form an
intermediate enzyme-substrate (ES) complex
E + S
k1
k-1
ES
k2
E + P
k-2
k1, k-1, k2, k-2 - rate constant - indicate the speed
or efficiency of a reaction
Rate of Catalysis
- At a fixed enzyme concentration [E],
the initial velocity Vo is almost linearly
proportional to substrate concentration
[S] when [S] is small but is nearly
independent of [S] when [S] is large
- Rate rises linearly as [S] increases and
then levels off at high [S] (saturated)
The Michaelis-Menten Equation
The basic equation derived by Michaelis and Menten to explain
enzyme-catalyzed reactions is
Vmax[S]
vo =
Km + [S]
Km - Michaelis constant;
Vo – initial velocity caused by substrate concentration,
[S];
Vmax – maximum velocity
Effect of enzyme concentration [E]
on velocity (v)
In fixed, saturating
[S], the higher the
concentration of
enzyme, the greater
the initial reaction
rate
This relationship will
hold as long as there
is enough substrate
present
Enzyme inhibition
In a tissue and cell different chemical agents
(metabolites, substrate analogs, toxins,
drugs, metal complexes etc) can inhibit the
enzyme activity
Inhibitor (I) binds to an enzyme and prevents
the formation of ES complex or breakdown it to
E+P
Reversible and irreversible
inhibitors
Reversible inhibitors – after combining with
enzyme (EI complex is formed) can rapidly
dissociate
Enzyme is inactive only when bound to inhibitor
EI complex is held together by weak,
noncovalent interaction
Three basic types of reversible inhibition:
Competitive, Uncompetitive, Noncompetitive
Reversible inhibition
Competitive inhibition
•Inhibitor has a structure similar to the substrate
thus can bind to the same active site
•The enzyme cannot differentiate between the
two compounds
•When inhibitor binds, prevents the substrate
from binding
•Inhibitor can be released by increasing substrate
concentration
Competitive inhibition
Example of
competitive
inhibition
Benzamidine
competes with
arginine for binding
to trypsin
Noncompetitive inhibition
• Binds to an enzyme site different from the active
site
• Inhibitor and substrate can bind enzyme at the same
time
•Cannot be overcome by increasing the substrate
concentration
Uncompetitive inhibition
• Uncompetitive inhibitors bind to ES not to free E
• This type of inhibition usually only occurs in
multisubstrate reactions
Irreversible Enzyme Inhibition
very slow dissociation of EI complex
Tightly bound through covalent or noncovalent
interactions
Irreversible inhibitors
•group-specific reagents
•substrate analogs
•suicide inhibitors
Group-specific reagents
–react with specific R groups of amino acids
Substrate analogs
–structurally similar to the substrate for the
enzyme
-covalently modify active site residues
Suicide inhibitors
•Inhibitor binds as a substrate and is initially
processed by the normal catalytic mechanism
•It then generates a chemically reactive
intermediate that inactivates the enzyme
through covalent modification
•Suicide because enzyme participates in its
own irreversible inhibition