Enzyme Kinetics

Enzyme Kinetics

• Enzyme Kinetics – Quantitative measurement of the rates

of enzyme catalyzed reactions
&
• The systematic study of factors that affect these rates

• Enzyme kinetics began in 1902 when Adrina Brown

reported an investigation of the rate of hydrolysis of sucrose
as catalyzed by the yeast enzyme inveratase.

• Brown demonstrated – when sucrose concentration is

much higher than that of the enzyme, reaction rate
becomes independent of sucrose concentration
Enzyme Kinetics

• Brown proposal – overall reaction is composed of two

elementary reactions in which the substrate forms a
complex with the enzyme that subsequently decomposes to
products and enzymes.

k1 k2
E + S ES P+E
k-1
• Here E, S, ES and P symbolize the enzyme, substrate,
enzyme-substrate complex and products
Enzyme Kinetics

• According to this model

• When the substrate concentration becomes high enough
to entirely convert the enzyme to the ES form, the
second step of the reaction becomes rate limiting step.
• The overall reaction rate becomes insensitive to further
increase in substrate concentration.

• The general expression of the velocity (rate) of this

reaction is

d [ P]
v= = k [ ES ]
dt 2
Enzyme Kinetics

• The overall rate of production of [ES] – Difference between the

rates of elementary reactions leading to its appearance and
those resulting in its disappearance.
K1
k2
E+S ES   → P + E
K-1
d [ ES ]
= k1[ E ][ S ] − k −1[ ES ] − k 2 [ ES ]
dt
• At this point, an assumption is required to achieve an analytical
solution.
• The rapid equilibrium assumption
• Michaelis - Menten Approach.
• The steady-state assumption.
• Briggs and Haldane Approach.
Michaelis - Menten Approach
The rapid equilibrium assumption:
• Assumes a rapid equilibrium between the
enzyme and substrate to form an [ES] complex.
K1 k2
E+S ES   → P + E
K-1

k1[ E ][ S ] = k −1[ ES ]
• The equilibrium constant Km can be expressed by
the following equation in a dilute system.
k −1 [ E ][ S ]
Km = =
k1 [ ES ]
Michaelis - Menten Approach
• Since the enzyme is not consumed, the
conservation equation on the enzyme yields
[ E ] = [ E0 ] − [ ES ]
• Then rearrange the equilibrium constant
equation
k −1 [ E ][ S ] [ E ][ S ]
Km = = [ ES ] ==
k1 [ ES ] Km
• Substituting [E] in the above equation with
enzyme mass conservation equation
([ E0 ] − [ ES ])[ S ]
[ ES ] ==
Km
Michaelis - Menten Approach
([ E0 ] − [ ES ])[ S ]
[ ES ] ==
Km
[ ES ]Km == [ E0 ][ S ] − [ ES ][ S ]

[ ES ]Km + [ ES ][ S ] == [ E0 ][ S ]

[ ES ]( Km + [ S ]) == [ E0 ][ S ]

[ E0 ][ S ]
[ ES ] ==
Km + [ S ]
Michaelis - Menten Approach
• Then the rate of production formation v can
be expressed in terms of [S]
d [ P] k 2 [ E0 ][ S ] Vmax [ S ]
v= = k 2 [ ES ] = =
dt Km + [ S ] Km + [ S ]
• Where Vmax = k 2 [ E0 ]
Steady State Assumption (SSA)
• Progress curve for the
components of a simple
michaelis-Menten
reaction
• Except the transition
phase of the reaction
(before shaded block)
[ES] remains constant
until the substrate is
nearly exhausted.
• Hence synthesis of ES
must equals to its
consumption over the
course of reaction i.e. ES
maintain steady state
SSA and Rate Equation

•Now: Base on steady state assumption, d[ES]/dt = 0

•d[ES]/dt = k1[E][S] –k-1[ES] – k2[ES] = 0

(steady state assumption)

•solve for [ES] (do some algebra)

•[ES] = [E][S] k1/(k-1 + k2)

•Define KM (Michealis Constant)

•KM = (k-1 + k2)/k1 => [ES] = [E][S]/KM
 SSA and Rate Equation
• Substitute [ E ] = [ E0 ] − [ ES ] in KM = [E][S]/[ES]
([ E 0 ] − [ ES ])[ S ]
Km =
[ ES ]
Km[ ES ] = ([ E 0 ] − [ ES ])[ S ]; [ ES ]Km == [ E0 ][ S ] − [ ES ][ S ]

[ ES ]Km + [ ES ][ S ] == [ E0 ][ S ]
[ ES ]( Km + [ S ]) == [ E0 ][ S ]
[ E0 ][ S ]
[ ES ] ==
Km + [ S ]
SSA lead to Michaelis - Menten
• Then the rate of production formation v can
be expressed in terms of [S]
d [ P] k 2 [ E0 ][ S ] Vmax [ S ]
v= = k 2 [ ES ] = =
dt Km + [ S ] Km + [ S ]
• Where Vmax = k 2 [ E0 ]

• Michaelis Menten Equation

Vmax [ S ]
v=
Km + [ S ]
Michaelis Menten Equation
• Michaelis-Menten equation, the rate equation for
a one-substrate enzyme-catalyzed reaction.

• It is a statement of the quantitative relationship

between the initial velocity V0, the maximum velocity
Vmax, and the initial substrate concentration [S], all
related through the Michaelis constant Km.
Michaelis Menten Equation
• Numerical relationship emerges from the Michaelis-
Menten equation in the special case when V0 is
exactly one-half of Vmax

• On dividing by Vmax we obtained

• Solving for Km, we get Km + [S] = 2[S] 1

v 0 = Vmax
Km = [S] when 2
Km
• KM is the substrate concentration required to reach
half-maximal velocity (vmax/2).
• KM is a measure
of a substrate’s
affinity for the
enzyme.
• A small KM
means the
substrate binds
tightly to the
enzyme and
saturates the
Vmax
• Considering the total enzyme concentration the
maximal rate, that the enzyme can attain is Vmax,.
• Vmax is equal to the product of the catalytic rate
constant (kcat) and the concentration of the enzyme.
• The Michaelis-Menten equation can then be
rewritten as V= Kcat [Enzyme] [S] / (Km + [S]).
• Kcat is equal to K2, and it measures the number of
substrate molecules "turned over" by enzyme per
second.
• The higher the Kcat is, the more substrates get
turned over in one second.
Michaelis-Menten Kinetics
Features of Michaelis-Menten

• Assumes the formation of Enzyme substrate

complex
• Assumes that the ES complex is in rapid equilibrium
with free enzyme
• Breakdown of ES to form products assumed to be
slower than Vmax [ S ]
1. Formation of ES and v0 =
K m + [S ]
2. Breakdown of ES to reform E and S
Michaelis-Menten Kinetics
[ ES ]
KA =
[ E ][ S ]
• KA is an equilibrium association constant (units: M -1)
[ E ][ S ]
KD =
[ ES ]

• KD is an equilibrium dissociation constant (units: M)

• Tight binding implies a low dissociation constant

and a high association constant
Transformations of the Michaelis-Menten
Equation: The Double-Reciprocal Plot
• The direct measurement of the numeric value of Vmax
and therefore the calculation of Km often requires
impractically high concentrations of substrate to
achieve saturating conditions

• The Michaelis-Menten equation

Vmax [ S ]
can be algebraically transformed v0 =
into equations that are more useful K m + [S ]
in plotting experimental data.
Lineweaver-Burk Equation
• Starting with the MM equation Vmax [ S ]
v0 =
K m + [S ]
• Reciprocal of MM equation 1 Km 1
= +
v 0 Vmax [ S ] Vmax
1 K 1 1
• Lineweaver-Burk Equation =( m
) +
v 0 Vmax [ S ] Vmax

• Equation is the equation for a straight line, y = ax +

b, where y = 1/v0 and x = 1/[S].
Lineweaver-Burk Equation
• A plot of 1/v0 as y as a function of 1/[S] as x therefore
gives a straight line whose y intercept is 1/Vmax and
whose slope is Km/Vmax.
• Such a plot is called a
double reciprocal or
Lineweaver-Burk plot
• Setting the y term of equation
equal to zero and solving for
x reveals that the x intercept
is −1/Km
Lineweaver-Burk Equation
• 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]

• The double-reciprocal plot of enzyme reaction

rates is very useful in distinguishing between
certain types of enzymatic reaction
mechanisms.
Kinetics of Isosteric enzymes

• Isosteric enzymes
(with only one enzyme
conformation, 1), the
efficiency of
substrate binding
(dashed curve)
declines constantly
with increasing [A],
because the number
of free binding sites is
constantly decreasing.
Kinetics of allosteric enzymes
• Allosteric enzymes, the
binding efficiency initially
rises with increasing [A],
because the free enzyme
is present in a low-affinity
conformation (square
symbols), which is
gradually converted into a
higher-affinity form(round
symbols) as a result of
binding with A.
• It is only at high [A] values
that a lack of free binding
sites becomes noticeable
and the binding strength
decreases again.
Enzyme Kinetics - Factors
• The catalytic properties of enzymes, and
consequently their activity, are influenced by
numerous factors.
• These factors include
• Physical quantities (temperature, pressure),
• The chemical properties of the solution (pH value,
ionic strength),
• The concentrations of the relevant substrates,
cofactors, and inhibitors.
pH Dependency of Enzyme Activity
• Effect of enzymes is strongly dependent on the pH

• Activity is plotted against pH, a bell-shaped curve is

usually obtained

• Bell shape of the activity–pH profile results from the

fact that amino acid residues with ionizable groups in
the side chain are essential for catalysis.
pH Dependency of Enzyme Activity
• a basic group B (pKa = 8),
which has to be protonated
in order to become active.
• a second acidic amino acid
AH (pKa = 6), which is only
active in a dissociated state.
• At the optimum pH of 7,
around 90% of both groups
are present in the active form
• at higher and lower values,
one or the other of the
groups increasingly passes
into the inactive state.
Temperature Dependency of Enzyme
Activity
• The temperature
dependency of enzymatic
activity is usually
asymmetric.
• With increasing temperature,
the increased thermal
movement of the molecules
initially leads to a rate
acceleration
• At a certain temperature, the
enzyme then becomes
unstable, and its activity is
lost within a narrow
temperature difference as a
result of denaturation
Bisubstrate Kinetics
• Most reactions in biological systems usually include two
substrates and two products A + B -> P + Q.

• In bisubstrate reactions transfer of a functional group, such as a

phosphoryl or an ammonium group, from one substrate to the
other

• In oxidation-reduction reactions, electrons are transferred

between substrates

• Multiple substrate reactions can be divided into two classes:

sequential displacement and double displacement.
Bisubstrate Kinetics
Sequential Displacement
• In the sequential mechanism, all substrates must bind to the
enzyme before any product is released.

• Sequential mechanisms are of two types: ordered, in which the

substrates bind the enzyme in a defined sequence, and
random.

• Many enzymes that have NAD+ or NADH as a substrate exhibit

the sequential ordered mechanism

• Lactate dehydrogenase reduces pyruvate to lactate while

oxidizing NADH to NAD+.
Bisubstrate Kinetics
Sequential Displacement
• In the ordered sequential mechanism, the coenzyme always
binds first and the lactate is always released first.
Bisubstrate Kinetics
Sequential Displacement
• Random sequential mechanism, the order of addition of
substrates and release of products is random.

• E.g. formation of phosphocreatine and ADP from ATP and

creatine, a reaction catalyzed by creatine kinase

• Sequential random reactions can also be depicted in the

notation.
Bisubstrate Kinetics – Ping-Pong
• In double-displacement, or
Ping-Pong, reactions, one or
more products are released
before all substrates bind the
enzyme.
• Mechanisms in which the
first substrate A is bound and
immediately cleaved.
• A part of this substrate
remains bound to the
enzyme, and is then
transferred to the second
substrate B after the first
product C has been
released. – Ping-Pong
Bisubstrate Kinetics – Ping-Pong
• The enzyme aspartate aminotransferase catalyzes the transfer
of an amino group from aspartate to a-ketoglutarate.

• After aspartate binds to the enzyme, the enzyme removes aspartate's

amino group to form the substituted enzyme intermediate.

• The first product, oxaloacetate, subsequently departs.

• The second substrate, a-ketoglutarate, binds to the enzyme, accepts the

amino group from the modified enzyme, and is then released as the final
product, glutamate.