ENZYME KINETICS
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BIOCHEMISTY
BINITA RANI
ASSOCIATE PROFESSOR (DAIRY CHEMISTRY)
FACULTY OF DAIRY TECHNOLOGY
S.G.I.D.T., BVC CAMPUS,
P.O.- BVC, DIST.-PATNA-800014
Course No.-DTC-111, Credit Hours – 2 (1+1)
ENZYME KINETICS
In the
absence of enzyme =>
conversion of S to P is
slow
and
uncontrolled
.
In presence of a
specific enzyme =>
S is converted
swiftly and
specifically
to
product
.
Enzyme is
specific
=>
it will not convert A to B or X to Y.
Enzymes also provide
=>
rate acceleration
.
On comparing rate of a
chemical reaction in solution =>
with =>
rate of the same reaction with the
reactants bound to enzyme
=>
enzyme reaction
will occur up to
1014 times faster
.
A unit is
=>
amount of enzyme
=>
that will catalyze the
conversion of
1 μ mol of substrate
to
product
in
1 min
under a given set of conditions.
Units of enzyme can be converted to
=>
milligrams of
enzyme by a
conversion factor
called
=>
specific activity
.
Specific activity
is
=>
amount of enzyme activity per
milligram of protein (micromoles of product formed / minute
/ milligram of protein, or units per milligram).
For a pure enzyme
=>
under a defined set of conditions
=>
the
specific activity
is a
constant
; however, different enzymes
have
=>
different specific activities
.
An
enzyme assay
is
=>
act of measuring
how
fast
a given
(or unknown) amount of enzyme will convert substrate to
product -
act of measuring a velocity
.
Velocity
(rate, v, activity, d[P]/dt, d[S]/dt) is
=>
how fast an
enzyme converts
substrate to product
=>
the amount of
substrate consumed, or product formed
per unit time
.
Units are
micromoles per minute
(μmol/min) units.
The Michaelis-Menten Equation
primary function of enzymes =>
to
enhance rates
of reactions
=>
so that they are compatible with
=>
needs of the organism.
To understand how enzymes function
=>
we need a
kinetic description of their activity.
For many enzymes
=>
the rate of catalysis
V0
=>
which is
defined as
number of moles of product formed per second
=>
varies with
substrate concentration
[S].
The rate of catalysis
=>
rises linearly as substrate
concentration increases
and
=>
then
begins to level off
and
approach a maximum at
higher substrate concentrations
.
Effect of substrate concentration on the initial velocity of an enzyme-catalyzed reaction
The extent of
product formation
is determined
=>
as a
function of time =>
for a
series of substrate concentrations
.
In each case
=>
amount of
product formed
increases with time
=>
although eventually
=>
a time is reached when
=>
there is
no net change in the concentration
of S or P.
Enzyme is still actively converting
=>
substrate into product
and visa versa
=>
but
reaction equilibrium
has been attained.
We define
V0
as
=>
rate
of increase in product with time
when
[P] is low
=>
that is
=>
at times close to
zero
(hence, V0).
Thus,
V0
is determined for each substrate concentration
=>
by
measuring the
rate of product formation at early times
before
P accumulates .
We begin our
kinetic examination
of
enzyme activity
with
the graph shown in Figure.
At a
fixed concentration
of enzyme
=>
V 0 is almost
linearly proportional
to [S] when [S] is
small
but
is nearly
independent
of [S] when [S] is
large
.
In 1913, Leonor Michaelis and Maud Menten proposed
=>
a simple model to
=>
account for these kinetic
characteristics.
The
critical feature
in their treatment is
=>
that a specific
ES complex
is a necessary intermediate in catalysis.
The model proposed
=>
is the simplest one that accounts
for the kinetic properties of many enzymes.
To simplify matters
=>
we will work under
steady-state
assumption.
In a steady state
=>
the concentrations of intermediates, in
this case
[ES] =>
stay the same
even if
=>
concentrations of
starting materials and products are changing.
This occurs
=>
when the rates of formation and breakdown of
the
ES complex
are
equal
.
Setting the right-hand sides of equations 3 and 4 equal gives
Now let us examine the numerator of equation 8.
The concentration of uncombined substrate [S] is very
nearly
=>
equal to the total substrate concentration
=>
provided that the concentration of enzyme is much lower
than that of substrate.
The concentration of
uncombined enzyme
[E] is equal to
the
total enzyme concentration
[E]T minus the
concentration of the
ES complex.
At very
low substrate concentration =>
when [S] is much less
than K M, V0 = (V max/K M)[S]
=>
that is
=>
the rate is
directly proportional to the substrate concentration
.
At
high substrate concentration =>
when [S] is much greater
than K M, V0 = V max
=>
that is
=>
the
rate is maximal =>
independent of substrate concentration
.
The meaning of K M is evident from equation 15.
When V0 = V max/2
Then [S] = KM
Thus,
K M
is equal to the substrate concentration at which
=>
the
reaction rate is half its maximal value
.
Km
is an
important characteristic
of an
enzyme-catalyzed
reaction
and is significant for
=>
its biological function
.
For most enzymes,
Km
lies between
10-1 and 10-7 M
.
The Significance of Km and Vmax Values
Km
value for an enzyme depends on
=>
particular substrate
and on
environmental conditions => pH, temperature, and ionic strength.
Km
is
=> concentration of substrate
at which half the active sites are
filled. Thus
=>
Km provides a measure of the substrate concentration
required for
=>
significant catalysis
to occur.
Km
is equal to
=> dissociation constant of ES complex
if k2 is
<
k-1 .
High Km indicates
=>
weak
binding
=>
a
low Km
indicates
strong
binding
. Km indicates
affinity of ES complex
only when k- 1 is
>
k2.
The maximal rate
=>
V max =>
reveals the turnover number of an
enzyme
=>
i.e. the number of substrate molecules converted into
product by an enzyme molecule in a unit time
=>
when enzyme is
fully saturated with substrate.
The Double-Reciprocal Plot
Michaelis-Menten equation
can be algebraically transformed into
equations that are more useful in
plotting experimental data
This
form of Michaelis-Menten equation
is called
=>
Lineweaver-Burk equation
.
For enzymes obeying the Michaelis-Menten relationship
=>
a
plot of
1/V0
versus
1/[S]
yields
=>
a straight line.
This line has a slope of
Km/Vmax =>
an intercept of
1/Vmax
on
the
1/V0
axis, and
=>
an intercept of -1/Km on the 1/[S] axis.
The Double-
Reciprocal
Plot
The double-reciprocal presentation
=>
also called a
Lineweaver-Burk plot
=>
has the great advantage of
allowing a
more accurate determination of => Vmax
, which
can only be approximated from
=>
a simple plot of
V0
versus [S].
Double-reciprocal plot
of enzyme reaction rates is very
useful in
=>
distinguishing between certain types of
enzymatic reaction
mechanisms
and
=>
in analyzing
enzyme
inhibition
.
THANKS
In enzyme kinetics, the presence of specific enzymes accelerates reactions, with substrate conversion catalyzed swiftly and specifically. Enzyme units and specific activity play critical roles, impacting reaction rates and product formation. The Michaelis-Menten Equation explains enzyme behavior, with catalysis rates varying with substrate concentration. Explore the impact of substrate concentration on reaction velocity and the time-dependent product formation.
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Presentation Transcript
BIOCHEMISTY Course No.-DTC-111, Credit Hours 2 (1+1) ENZYME KINETICS BINITA RANI ASSOCIATE PROFESSOR (DAIRY CHEMISTRY) FACULTY OF DAIRY TECHNOLOGY S.G.I.D.T., BVC CAMPUS, P.O.- BVC, DIST.-PATNA-800014
In the absence of enzyme => conversion of S to P is slow and uncontrolled. In presence of a specific enzyme => S is converted swiftly and specifically to product. Enzyme is specific => it will not convert A to B or X to Y. Enzymes also provide => rate acceleration. On comparing rate of a chemical reaction in solution => with => rate of the same reaction with the reactants bound to enzyme =>enzyme reaction will occur up to 1014 times faster.
A unit is => amount of enzyme => that will catalyze the conversion of 1 mol of substrate to product in 1 min under a given set of conditions. Units of enzyme can be converted to => milligrams of enzyme by a conversion factor called => specific activity. Specific activity is => amount of enzyme activity per milligram of protein (micromoles of product formed / minute / milligram of protein, or units per milligram).
For a pure enzyme => under a defined set of conditions => the specific activity is a constant; however, different enzymes have => different specific activities. An enzyme assay is => act of measuring how fast a given (or unknown) amount of enzyme will convert substrate to product - act of measuring a velocity. Velocity (rate, v, activity, d[P]/dt, d[S]/dt) is => how fast an enzyme converts substrate to product => the amount of substrate consumed, or product formed per unit time. Units are micromoles per minute ( mol/min) units.
The Michaelis-Menten Equation primary function of enzymes => to enhance rates of reactions => so that they are compatible with => needs of the organism. To understand how enzymes function => we need a kinetic description of their activity. For many enzymes => the rate of catalysis V0 => which is defined as number of moles of product formed per second => varies with substrate concentration [S]. The rate of catalysis => rises linearly as substrate concentration increases and => then begins to level off and approach a maximum at higher substrate concentrations.
Effect of substrate concentration on the initial velocity of an enzyme-catalyzed reaction
The extent of product formation is determined => as a function of time => for a series of substrate concentrations. In each case => amount of product formed increases with time => although eventually => a time is reached when => there is no net change in the concentration of S or P. Enzyme is still actively converting => substrate into product and visa versa => but reaction equilibrium has been attained. We define V0 as => rate of increase in product with time when [P] is low => that is => at times close to zero (hence, V0). Thus, V0 is determined for each substrate concentration => by measuring the rate of product formation at early times before P accumulates .
We begin our kinetic examination of enzyme activity with the graph shown in Figure. At a fixed concentration of enzyme => V 0 is almost linearly proportional to [S] when [S] is small but is nearly independent of [S] when [S] is large. In 1913, Leonor Michaelis and Maud Menten proposed => a simple model to => account for these kinetic characteristics. The critical feature in their treatment is => that a specific ES complex is a necessary intermediate in catalysis. The model proposed => is the simplest one that accounts for the kinetic properties of many enzymes.
To simplify matters => we will work under steady-state assumption. In a steady state => the concentrations of intermediates, in this case [ES] => stay the same even if => concentrations of starting materials and products are changing. This occurs => when the rates of formation and breakdown of the ES complex are equal. Setting the right-hand sides of equations 3 and 4 equal gives
Now let us examine the numerator of equation 8. The concentration of uncombined substrate [S] is very nearly => equal to the total substrate concentration => provided that the concentration of enzyme is much lower than that of substrate. The concentration of uncombined enzyme [E] is equal to the total enzyme concentration [E]T minus the concentration of the ES complex.
At very low substrate concentration => when [S] is much less than K M, V0 = (V max/K M)[S] => that is => the rate is directly proportional to the substrate concentration. At high substrate concentration => when [S] is much greater than K M, V0 = V max => that is => the rate is maximal => independent of substrate concentration. The meaning of K M is evident from equation 15. When V0 = V max/2 Then [S] = KM Thus, K M is equal to the substrate concentration at which => the reaction rate is half its maximal value. Km is an important characteristic of an enzyme-catalyzed reaction and is significant for => its biological function. For most enzymes, Kmlies between 10-1 and 10-7 M.
The Significance of Km and Vmax Values Km value for an enzyme depends on => particular substrate and on environmental conditions => pH, temperature, and ionic strength. Km is => concentration of substrate at which half the active sites are filled. Thus => Km provides a measure of the substrate concentration required for => significant catalysis to occur. Km is equal to => dissociation constant of ES complex if k2 is < k-1 . High Km indicates => weak binding => a low Km indicates strong binding. Km indicates affinity of ES complex only when k- 1 is > k2. The maximal rate => V max => reveals the turnover number of an enzyme => i.e. the number of substrate molecules converted into product by an enzyme molecule in a unit time => when enzyme is fully saturated with substrate.
The Double-Reciprocal Plot Michaelis-Menten equation can be algebraically transformed into equations that are more useful in plotting experimental data
This form of Michaelis-Menten equation is called => Lineweaver-Burk equation. For enzymes obeying the Michaelis-Menten relationship => a plot of 1/V0 versus 1/[S] yields => a straight line. This line has a slope of Km/Vmax => an intercept of 1/Vmax on the 1/V0 axis, and => an intercept of -1/Km on the 1/[S] axis. The Double- Reciprocal Plot
The double-reciprocal presentation => also called a Lineweaver-Burk plot => has the great advantage of allowing a more accurate determination of => Vmax, which can only be approximated from => a simple plot of V0 versus [S]. Double-reciprocal plot of enzyme reaction rates is very useful in => distinguishing between certain types of enzymatic reaction mechanisms and => in analyzing enzyme inhibition.
