 INTRODUCTION

 HISTORY
 ENZYME KINETICS
 MICHAELIS MENTEN APPROACH
 STEADY STATE EQUATION
 EFFECTS OF INHIBITORS
 MODIFICATION TO MM EQUATION
 FACTORS EFFECTING ENZYME KINETICS
 REFERENCES
 An enzyme is a biocatalyst that increase the rate
of chemical reaction without itself being
changed in the overall process.

 Study of reaction rates is an important tool to

investigate the chemical mechanism of catalysis.

 Kinetics study provide information on substrate

and product affinity.

 Enzyme kinetics include the study of the

chemical reactions that are catalyzed by the
enzyme.

• Enzyme Kinetics – Quantitative measurement of
the rates of enzyme catalyzed reactions and the
systematic study of factors that affect these rates

Kinetics studies measure reaction rates and the

affinity of enzyme for substrates and inhibitors
 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

• In 1913 enzyme catalysed reaction was devised by Leonor

Michaelis and Maud menten.

• Briggs and haland revised Michaelis Menten model in 1925.

• 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
• 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
• 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
k 2
E+S ES ⎯⎯→P + E
K-1
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.
 K1 k 2
E+S ES ⎯⎯→P + E
K-1
 The rate of formation of [ES] complex at
any time ‘t’
 t = K +1 [E] [S]
 Also at time ‘t’rate of breakdown of [ES]
back to [E] and [S].
  t = K-1 [ES]
 As per MM assumption equilibrium
 K+1 [E] [S] = K-1 [ES]
 [E] [S]/ [ES] = K-1 /K+1 =Km (1)
 Km is the dissociation constant of [ES]
 Total enzyme present [Et] must be the sum of [E]
and [ES]
 [E] = [E]t - [ES] (2)
• Since the enzyme is not consumed, the
conservation equation on the enzyme yields
[E] = [Et] −[ES ]
• Substituting [E] in the equation 1 with
enzyme mass conservation equation
([Et ] −[ES ])[S]
Km =
[ES]
 ([Et] −[ES ])[S]
[ES ] ==
Km
[ES ]Km == [Et ][S ] −[ES ][S ]

[ES ]Km +[ES ][S ] == [Et ][S ]

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

[Et ][S]
[ES ] ==
Km +[S ]
 Then the rate of appearance of product 'v' is
proportional to the concentration of the [ES]

V= Km × [ES]
The maximum reaction rate Vmax will be occur
at a point where total enzyme bound to the
substrate. Then Vmax,
Vmax = Km x [Et]
V/ Vmax = Km X [Et]

V =Vmax [S]
Km + [S]
 The interpretation of Michaelis Menten
model were refined by Briggs and Haldane by
an assumption termed the steady state
assumption.

 This hypothesis states that the concentration

of enzyme substrate complex remains
constant through much of the reaction.

 When the enzyme and substrate are mixed,

the concentration [ES] will rise rapidly from
zero to a so called steady state level.
• 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
The rate of formation of [ES] at any time ‘t’ is
t =k+1 [E] [S]
The rate of breakdown of [ES] at this time is
t = k-1 [ES] + k2 [ES]
As per assumption equilibrium was made
between
k+1[E][S] = k-1 [ES] +k2 [ES]
k+1[E][S] = [ES] (K-1 +K2)
[ES] [S] = k-1 + k2 = km
[ES] k+1
km is the disociation constant of [ES] complex
• Substitute [E] = [Et] −[ES ] in KM= [E][S]/[ES]
([Et ] −[ES ])[S]
Km =
[ES]
Km[ES] = ([Et ] −[ES ])[S]
[ES ]Km = [Et][S] −[ES ][S ]
[ES ]Km +[ES ][S ] = [Et ][S ]
[ES ](Km +[S ]) = [Et ][S ]
[Et ][S]
[ES ] =
Km +[S ]
 The rate of appearance of product (V) is directly proportional
to the concentration of [ES] complex.

V = km × [ES]
The maximum reaction rate (Vmax) will occur at a point where the total
enzyme concentration [Et] is bound to the substrate [S] then the maximum
concentration of [ES] complex will be equal to the total enzyme concentration.

Vmax = km × [Et]

V = [ES] = [S]
Vmax [ Et] [S] + km

V = Vmax [S]
km + [S]

Vmax = Vmax [S]

2 km + [S]
• 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.
• Numerical relationship emerges from the Michaelis-
Menten equation in the special case when V0 is
exactly one-half of Vmax

• On dividing by Vmaxwe obtained

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

v 0 = V max
Km = [S] when 2
• KMis 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
• 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 [E][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.
• 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 V [S ]
1. Formation of ES and v0 = max

K m + [S ]
2. Breakdown of ES to reform E and S
• The direct measurement of the numeric value of Vmax
and therefore the calculation of Kmoften requires
impractically high concentrations of substrate to
achieve saturating conditions

• The Michaelis-Menten equation v0 = V

max [S]
can be algebraically transformed K m + [S ]
into equations that are more useful
in plotting experimental data.
• Starting with the MM equation Vmax [S]
v0 =
K m + [S ]
1 K 1
• Reciprocal of MM equation = m
+
v0 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].
• 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.
• 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.
 • 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.
 The temperature dependency of enzymatic
activity is usually asymmetric.
 With increasing temperature the increased
thermal movement of 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
result of denaturation.
 Jeremy M. Berg, John L.Tymaczko, Lubert
Stryer, 2002, Biochemistry, fifth edition, New
York, Printed in the United States of America.
 Pranav Kumar, Usha Mina, 2017, Life Science
fundamentals and practice I, sixth edition, New
Delhi, India, Pathfinder publication.
 Micheal M. Cox, David L. Nelson, Albert L.
Lehniger, 1970, Principles of biochemistry,
fifth edition, Replica press Pvt.Ltd.
Thank you…