FREE ENERGY OF ENZYME

REACTIONS. STEADY STATE

KINETICS. MICHAELIS-MENTON,
LINEWEAVER–BURK, EDDE-
HOFSTEE AND HANES-WOOLF
EQUATIONS
 INTRODUCTION

Enzymes are biological catalysts that increases the rate of

biochemical reactions taking place within living cells without
themselves suffering any overall change.
They are one of the most important biomolecules in universe on
which is very thread of life. Every bioreactions are carried out by
some enzymes, even though they are only biocatalysts and used to
speed up the reaction, all life in universe have evolved into a state
that their bioreactions cannot be carried out in absence of the
enzyme.
Properties of enzymes:-
 The most important property of enymes is it is produced within cells,
through translation of mRNA which is produced by transcription of
DNA.
They are highly specific in nature to their substrate
They are thermolabile and pH labile
• Enzymes does not modify:
• – The equilibrium constant and
• – The Gibbs free energy change of a reaction.
BASIC MECHANISM OF
ENZYMATIC REACTION
 FREE ENRGY AND GIBBS ENERGY

• A process will only happen spontaneously, without added energy, if it increases

the entropy of the universe as a whole ,this is the Second law of
thermodynamics..
• The Gibbs free energy (G) of a system is a measure of the amount of usable energy
(energy that can do work) in that system. The change in Gibbs free energy during a
reaction provides useful information about the reaction's energetics and spontaneity
ΔG=ΔH−TΔS
∆H is the enthalpy change, ∆S is the entropy change of the system
Temperature (T).
 Gibbs free energy
(G)

EXERGONIC
REACTIONS ΔG = 0,a chemical ENDERGONIC
reaction is REACTIONS
Reactions with a in equilibrium, the
negative ∆G release rates of forward and A positive ∆G
energy, proceed reverse reactions need energy
without an energy are equal, so there is input(non-
input (are no net change.
spontaneous) spontaneous).
• Cells couple exergonic reactions to endergonic reactions so that
the net free energy change is negative
• cells accomplish endergonic reactions such as active transport, cell
movement or protein synthesis by tapping the energy of ATP
hydrolysis:
• the sign of ΔG (negative or positive) determines the direction that the
reaction will go spontaneously, the magnitude of ΔG does not predict
how fast the reaction will go.
• The rate of the reaction is determined by the activation energy (the
energy required to attain the transition state) barrier Ea:
• Peak of the graph suggests the intermediate stage ES complex which
is interconvertible in reaction and can go in either direction.
• And it should be understood from the graph, reactions with most
activation energy Ea will proceed further.
• The addition of enzyme provides an alternative transition state with
lower activation energy. In the presence of the enzyme, a much
higher percentage of molecules (X or Y) can acquire enough energy
to attain the transition state, so the reaction can go faster, in either
direction. It is done by attaching and binding to substrate later
converting it to product.
 ENZYME KINETICS
• Enzyme kinetics studies how the rate of enzyme-catalysed reactions
depends on the concentration of the compounds directly interacting
with the enzyme and what is the highest rate achievable by the
enzyme,how the rate of the catalysed reaction depends on the
temperature, pH, ionic strength etc. of the medium.
• The kinetics of enzyme-catalyzed reactions can be treated using any
one of the following approaches: –
• Steady-state approximation (SSA) or Briggs-Haldane Approach –
Rapid Equilibrium Approach (REA)
• We will focus more on SSA.
 ENZYME VELOCITY AND INFLUENCING
FACTORS
V0 is the rate of the reaction at the beginning of reaction when
enzyme starts to uitilise the substrate or bind to it and convert it to
product.
 The velocity or rate of reaction at time t is Vt.
The contact between enzyme and substrate is prerequisiste for the
enzyme activity. There are many factors that affect the rate of the
reaction:-
1.Concentration of the enzyme.
• [E] increases ,V of the reaction increases.
• Because as the enzyme amount increases more and more susbtrate is
utilized and more product is formed.
• This method is actually used in serum enzymes the diagnosis of
dieseases which is done by keeping all other parameters constant.
2.Effect of temperature.
Velocity V increases as the temperature T increases up to a
maximum and then declines. Giving the bell shaped curve.
It is because as T
increases , more and more
molecules will achieve
the Ea quickly and due to
more collision and
interaction of molecules
the reaction procced
faster.
The optimum temperature
of most enzymes is 350C-
400C. Exceptions are Taq
polymerase active at 950C
and plant urease active at
600C.
3. Effect of pH
• Increase in hydrogen ion concentration (pH) ion influences the
enzyme activity and a bell shaped curve is obtained.
It is because hydrogen ions
influence the enzyme activity by
altering the ionic charges on the
amino acids of enzymes at the
active site , substrate and ES
complex.
The optimum pH for most
enzymes is neutral around pH6-8
.exceptions are pepsin (1-2),
alkaline phosphatase(10-11).
4. Effect of the product concentration
• The accumulation of product concentration decreases reaction
velocity . for certain enzymes , the products combine with active site
of enzymes and form a loose complex , thus inhibit enzyme activity.
5. Effect of activators
• Some of the enzymes require inorganic metallic cations like Mg2+
,Mn2+,Zn2+, Ca2+ etc…. for optimum activity. These ions act as
activators for enzyme activity. Their low concentrations results in
decreased or no activity of enzyme activity.
6.Effect of substrate concentration.
• Rise in [S] increases the rate of enzymatic reaction. Within limited
range of substrate levels where the rectangular hyper bola is obtained
when rate of reactions is plotted against substrate concentration.
• This kinetic pattern was given by Victor Henrii following Wurtz’s
lead to propose in 1903 that the complex is needed for enzyme
catalysis.
• Higher [S] ,V0 increases by small amount. A point is reached beyond
which increases in V0 vanishingly small as increases . The plateau -
like V0 region is close to the maximum velocity.
• The idea of effect of substrate concentration was expanded by Leonor
Michaelis and Maud Menton in 1913 who proposed the the reaction
steps :-
The maximum initial velocity rate observed is Vmax
which is when all enzyme is in the ES complex form and
[E] is 0. This saturation effect is distinguishing
characteristic of enzymes and responsible plateau.
 STEADY STATE KINETICS
Proposed by G.E.Briggs and Haldane in 1925.
When the enzyme is mixed with a large amount of substrate, there is a
initial transient period called pre steady state.
During which ES concentration builds up and lasts just micro seconds.
The reaction quickly arrives in a state where a [ES] concentrations
remain approximately constant over time.
The product is generated at the same rate as the Substrate consumed
and ES concentration remain constant or steady.
The measured V0 generally reflects the steady state , even though V0 is
limited to the early part of the reaction and analysis of these rates is
referred to as steady state kinetics .
 MICHAELIS MENTON EQUATION
Proposed by Leonor Michaelis and Maud Menton in 1913 .
E + S← k1 ← →k2→ ES →k3→ E + P

This is Michaelis Menton equation , the rate equation for one

substrate enzyme cataylsed reaction. it is statement of quantitative
realtionship between initial velocity V0 and maximum velocity
Vmax. the initial substrate concentration [S] and related to Km
constant.
• An important numerical relationship emerges in special case when V0
is exactly one half of Vmax. (Vmax/2 )= Vmax[S]/(Km+[S])
on dividing the Vmax ,
½ = [S]/(Km+[S])
Solving for Km we get
Km + [S] = 2[S]
i.e., Km = [S]
• This is very useful in practical definition of Km is equivalent to
substrate concentration when V0 is one half of Vmax.
Significance
• The Michaelis-Menten model is used in a variety of
biochemical situations other than enzyme-substrate interaction,
including antigen-antibody binding, DNA-DNA hybridization,
and protein-protein interaction. It can be used to characterize a
generic biochemical reaction, in the same way that the
Langmuir equation can be used to model generic adsorption
of biomolecular species. When an empirical equation of this
form is applied to microbial growth.
 LINEWEAVER-BURK PLOT
Michaelis menton eq.
Vm [ S ]
v
Km  [ S ]
Double reciprocal plot
• Lineweaver-Burk plot gives good estimates on Vm but not
necessarily on Km (error relates with substrate conc)
• The Lineweaver-Burk plot, in with the inverse of the reaction rate,
1/r, is plotted against the inverse of the substrate concentration 1/[S].
• The Lineweaver–Burk plot (or double reciprocal plot) is a graphical
representation of the Lineweaver–Burk equation of enzyme kinetics,
described by Hans Lineweaver and Dean Burk in 1934 .
• The Lineweaver-Burk plot results in a straight line with the slope
equal to KM/k2[E]0 and y-intercept equal to 1/k2[E]0 which is 1/Vmax
Significance
The Lineweaver–Burk plot was widely used to determine important
terms in enzyme kinetics, such as Km and Vmax, before the wide
availability of powerful computers and non-linear regression
software. The y-intercept of such a graph is equivalent to the inverse
of Vmax the x-intercept of the graph represents −1/Km. It also gives
a quick, visual impression of the different forms of enzyme
inhibition and reaction mechanisms. When used for determining the
type of enzyme inhibition, the Lineweaver–Burk plot can distinguish
competitive, non-competitive and uncompetitive inhibitors.
 EADIE–HOFSTEE PLOT

Michaelis menton eq.

Vm [ S ]
v
Km  [ S ]
Rearranged MM equation

plot v versus v/[S] gives a line of

slope –Km and y-axis intercept of
Vm
• The Eadie–Hofstee plot is a graphical representation of enzyme
kinetics in which reaction rate is plotted as a function of the ratio
between rate and substrate concentration.
• A plot of v against v/[S] will hence yield Vmax as the y-intercept,
Vmax/Km as the x-intercept, and Km as the negative slope.
• Like other techniques that linearize the Michaelis–Menten equation,
the Eadie-Hofstee plot was used for rapid identification of important
kinetic terms like Km and Vmax.
• It is also more robust against error-prone data than the Lineweaver–
Burk plot, particularly because it gives equal weight to data points in
any range of substrate concentration or reaction rate.
 HANES–WOOLF PLOT
Michaelis menton eq.
Vm [ S ]
v
Km  [ S ]
Obtained by reciprocal and
multiplication [S] on both sides

This plot is used to determine Vm more accurately.

• The Hanes-Woolf plot is another method that linearises the
Michaelis-Menten equation, plotting [S] (x-axis) against [S]/ν (y-
axis).
• Just like the Lineweaver-Burk equation, the Hanes-Woolf
equation is of the form y = mx + c.