2 Factors Aff & KM, MMeq, DRP 2021

Factors affecting Enzyme
activity
z Dr. Rajlaxmi Sarangi
Professor
Dept. of Biochemistry
KIMS
z
Cont……………
 Factors affecting enzymatic activity
 Enzyme Specificity
 How Enzyme works – lowers activation energy
 Mechanism of enzyme action

Factors Affecting Enzyme Activity
Velocity or rate of enzyme reaction is the rate of change of substrate or
Formation of product per unit time.
Velocity is proportional to the concentration of reacting molecules
V  A B
K1
A +B C +D
K2
Forward reaction. R1 = K1 A B
Backward reaction R2 = K2 C D
AT EQuilibrium R1= R2
K1 A B = K2 C D
OR K1 C D Keq OR EQuilibrium

K2 A B Constant
Numerical value of constant calculated from concentration of substrate
and product.
Keq more than 1 forward reaction favored

Concentration of enzyme does not affect
the keq.
FACTORS INFLUENCING ENZYME ACTIVITY
1.Conc. Of Enzyme 2. Temperatur 3. pH 4. Substrate 5.product
1. Enzyme concentration
1.Rate of a reaction v is directly proportional to
enzyme concentration provided substrate is unlimited
ENZYME ACTIVITY
ENZYME CONCENTRATION
ENZYME ACTIVITY:
Micromoles of substrate converted to product per minute
expressed as one standard unit (international unit)
Katals (kat) - conversion of 1 mole of substrate / sec

2.EFFECT OF TEMPERATURE:
* Velocity increases as temperature increased
Increase in temp results in higher activation energy of the
molecule.
More collision and interaction between the enzyme and
substrate.
* Maximum velocity reached then falls
Temp at max velocity optimum temperature
* Further increase cause denaturation
EFFECT OF TEMPERATURE
100
TEMP C
The optimum temperature for most of The enzymes is between 40 – 45° C
3.EFFECT OF pH
OPTIMUM pH
100
%
HIGH
LOW
pH
Optimal activity between 5 - 9
Few enzyme active at low ph (pepsin) or
Higher ph (alkaline phophatase)
Related to the ionization of specific amino acids that
constitutes substrate binding site
Amino acid residues invoved in the catalysing the reaction
must be in the correct charge state to be functional
4.EFFECT OF SUBSTRATE CONCENTRATION
If the conc of substrate [s] is increased with all other
conditions are kept constant initial velocity  increases to
a maximum value Vmax and no further
Velocity plotted against [S] conc: Rectangular hyperbola
C
V MAX
1/2 V MAX
A
KM [S]
Part A:
At low substrate concentration, the velocity is directly
proportional to substrate conc
Part B:
The substrate concentration is not directly proportional to
the enzyme activity.
Part C:
the reaction rate is independent of substrate
concentration.
* At higher concentrations of substrate
all enzyme molecules are saturated
* increase in substrate does not affect

reaction velocity
* Maximum velocity obtained called vmax

DEFINITION OF KM: Michelis Constant
That substrate conc at which velocity of reaction is equal to
1/2 maximal velocity
* Km is independent of enzyme concentration
* Km value indicates affinity of enzyme for substrate higher

the km lower the affinity .
Km & Affinity are inversely related
MICHAELIS MENTEN EQUATION
The goal is to develop a relationship that allow the velocity of the
reaction to be corrleated with substrate concentration.
 = [VMAX ] [S]
[S] + Km
Vmax is unattainable
To find out different Enzyme Inhibition -Michalis Menten Eq.

k1 k3
E+S ES E+P
k2
K2+k3
Km =
k3
 = [S] [VMAX ]
[S] + Km
V= measured velocity
Vmax= maximum velocity
S= substrate concentration
Km= Michaelis – Menten constant
When [S] = Km
= [VMAX ] [S] = [VMAX ] [S] = [VMAX ]

Km+ [S] 2S 2
5.EFFECT OF PRODUCTS
When product conc is increased reaction slowed

or stopped
ENZYME ACTIVATION
Effect of
* metal ions
* co - enzymes
Increases enzyme activity
Specificity of enzymes
Active site/catalytic site –responsible for enzyme

specificity
-substrate binding site
-conc. Of substrate, affinity of active site for substrate are major
determinant of E-S interaction
Specificity is of three different types
3 Types of specificity
1. Stereospecity: enzymes act only on D-Carbohydrate & L-AA can not act on
other sterioisomer
. Eg. Enzymes acting on D-AA do not act on L-AA.
Amylase hydrolyse ⍺-glycosidic bond
Cellulase cleaves 𝝱-glycosidic bond
2. Reaction specificity :
Substrates can undergo many reactions.
Only one enzyme can catalyse one of the reactions.
Glu-6-p phosphohexose isomerase Fru-6-p
Glu-6 –p Glu-6 phosphatase Glu
Glu-6-p phosphoglucomutase Glu-1-P
3. Substrate specificity:
2 types of substrate specificity
*absolute specificity: urease catalyzes hydrolysis of only urea.
Glucokinase act only Liver glucose
Relative specificity: More than one substrate which are structurally related
Group dependent:
Eg. Trypsin, chymotrypsin. Cleaves by recognition of certain groups.
Bond specificity:
eg. Proteolytic enzymes, glycosidases, lipases acts on peptide bonds,
glycosidic bonds and ester bonds respectively.
Hexokinase acts on glucose, fructose,and mannose

Mechanism of enzyme action
1913: Michelis and Menten put forward the enzyme substrate
complex theory.
E+S E - S complex E+P
Theories to explain E-S interaction:

1. Lock & Key model: Emil fischer
2. Induced fit theory
3. Substrate strain theory
1. Lock & Key theory: - Emil Fischer
Three dimensional structure of the active site of the enzyme is
complimentary to the substrate.
Enzyme & substrate fit each other similar to lock and key.
This model gives rigid structure of the enzyme and fixed shape &
active site allows binding of only specific substrate.
Limitation: flexibility of enzyme is not considered.

cannot explain effects of allosteric ligands.
2. Koshland's induced fit model
Disadvantage of template model:
Active site-rigid & pre shaped. Highly rigid model
Advantage of koshland's model:
* flexibility of active site
*catalytic site not pre shaped to fit substrate and is not rigid.
* Substrate induces a conformational change
* Functional group of enzyme- Proper alignement orientation

for substrate binding catalysis
* In the absence of substrate, catalytic and substrate binding

groups are several bond distances apart
Induced fit model explain the action of Allosteric modulators

and competitive Inhibition of enzymes
3. Substrate strain theory
The interaction of substrate with enzyme
Induces a conformation changes in the enzyme &

substrate undergoes some strain
Formation of a stronger binding site by

Reposition of the amino acids ( breaking of existing bond
and formation of new bond).
Combination of induced fit & substrate strain is the

underlying mechanism of most of the enzymatic actions

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Factors affecting Enzyme

 Factors affecting enzymatic activity

 How Enzyme works – lowers activation energy

 Mechanism of enzyme action

OR K1 C D Keq OR EQuilibrium

* increase in substrate does not affect

* Maximum velocity obtained called vmax

* Km is independent of enzyme concentration

* Km value indicates affinity of enzyme for substrate higher

To find out different Enzyme Inhibition -Michalis Menten Eq.

= [VMAX ] [S] = [VMAX ] [S] = [VMAX ]

When product conc is increased reaction slowed

Active site/catalytic site –responsible for enzyme

Hexokinase acts on glucose, fructose,and mannose

Theories to explain E-S interaction:

Limitation: flexibility of enzyme is not considered.

* Functional group of enzyme- Proper alignement orientation

* In the absence of substrate, catalytic and substrate binding

Induced fit model explain the action of Allosteric modulators

The interaction of substrate with enzyme

Induces a conformation changes in the enzyme &

Formation of a stronger binding site by

Combination of induced fit & substrate strain is the

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