Introduction Kinetics and thermodynamicSG Thermodynamic in enzymatic reactions balanced equations in chemical reactions changes in free energy determine the direction & equilibrium state of chemical reactions the rates of reactions Factors effecting enzymatic activity (i) Enzyme concentration. (ii) Substrate concentration. (iii)Temperature (iv) pH. (v) Activators. (vi)Inhibitors Michaelis-menten equation CONCLUSIONS REFERENECES

1. Enzyme Kinetics And
Thermodynamic Analysis
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. CONTENTS
•Introduction
•Kinetics and thermodynamicSG
•Thermodynamic in enzymatic reactions
•balanced equations in chemical reactions
•changes in free energy determine the direction & equilibrium state of
chemical reactions
•the rates of reactions
•Factors effecting enzymatic activity
 (i) Enzyme concentration.
(ii) Substrate concentration.
(iii)Temperature
(iv) pH.
(v) Activators.
(vi)Inhibitors
•Michaelis-menten equation
•CONCLUSIONS
•REFERENECES

3. Introduction
The chemical reactions in all cells of living things operate in the presence of
biological catalysts called enzymes. Because a particular enzyme catalyzes
only one reaction, there are thousands of different enzymes in a cell catalyzing
thousands of different chemical reactions.
The substance changed or acted on by an enzyme is its substrate. The
products of a chemical reaction catalyzed by an enzyme are end products.

4. Kinetics and thermodynamic
The study of the rate at which an enzyme works is called enzyme kinetics. Let us
examine enzyme kinetics as a function of the concentration of substrate available
to the enzyme.
One can think of thermodynamics as the energy stored within a reaction, a
reactant, or a product.
Most often, we think of thermodynamics as the different forms of energy that are
converted every time a reaction excretes energy or uses energy to initiate itself.
Example: Water + Sugar - Thermodynamically-Stable

5. Thermodynamic in enzymatic reactions
BALANCED EQUATIONS IN CHEMICAL
REACTIONS
A balanced chemical equation lists the initial chemical species (substrates)
present and the new chemical species (products) formed for a particular chemical
reaction.
A+B P + Q ......... (1)
A+B  P+Q
...........(2)
Reactions for which thermodynamic factors strongly favor formation of the
products to which the arrow points often are represented with a single arrow as if
they were “irreversible”:

6. CHANGES IN FREE ENERGY DETERMINE THE DIRECTION &
EQUILIBRIUM STATE OF CHEMICAL REACTIONS
The Gibbs free energy change ΔG (also called either the free energy or Gibbs
energy) describes both the direction in which a chemical reaction will tend to
proceed and the concentrations of reactants and products that will be present
at equilibrium.
ΔG for a chemical reaction equals the sum of the free energies of formation of
the reaction products ΔGP minus the sum of the free energies of formation of
the substrates ΔGS. ΔG0 denotes the change in free energy that accompanies
transition from the standard state, one-molar concentrations of substrates and
products, to equilibrium.
ΔG0 = -RT In keq…..(3)

7. -----------------------------
-----------------
------------------
Enzyme are not involve
ΔG catalysis
ΔG uncatalysis
Enzyme involve
Free energy G
Reaction cordination
ES EP
Ground state
E+S ES EP E+P

8. THERATESOF REACTIONS
E +R −L E− R +L ………..(7)
E +R −L E... R.....L Δ GF............ (8)
E... R.....L E -R +L Δ GD ...............(9)
E +R −L E -R +L ΔG = ΔGF + ΔGD
..........(8-10)

9. •Factors effecting enzymatic activity
(1) Effect of E concentration on rate of Enzyme reaction
As E increases rate of reaction increases in a linear manner. However, some
deviations occur:
•upward curve
•downward curve
[E]
v

10. (ii) Effect of Substrate Concentration on the rate of E
catalysed reaction.
Mixed order reaction
Zero order reaction
1storder reaction
Km
Vmax
[S]
-----
--------------

11. [E]
v
•downward curve

12. (iii)Effect of Temperature on E catalysed reactions:
•At very low temperature e.g. O°C the rate of reaction may be almost zero.
•As temperature is increased rate of reaction increases.
•This occurs as the kinetic energy of the molecules increases.
•For every 10°C rise of temperature the rate is doubled. This is Q10or
Temperature Coefficient.
•But this occurs only up to a specific temperature which is known as Optimum
temperature.
•Beyond this temperature, the rate decreases sharply. This occurs as the enzyme
is denatured and the catalytic activity is lost.
•For most E, optimal temperature are at or slightly above those of the cell in
which the E occurs. Some E in bacteria which survive in hot springs have high
optimal temperature.

13. E is denature
Temperature
Optimum Temperature
Low kinetic energy
v
e.g. Human E:
~ 25-37°C
DNA polymerase in Taq Polymerase active
at upto100°C.[from Thermus aquitus.]
(iii)Effect of Temperature on E catalysed reactions

14. (iv)Effect of PH on E catalysed reaction.
When E actvityis measured at several pH values, optimal activityis generally
observed between pH values of 5-9. However, some E such as pepsin have low
pH optimum (! 2.0) which others have high pH optimum (e.g. Alkaline
Phosphatase(pH ~ 9.5).
The shape of pH activity curve is determined by the following:
(i)E is denatured at high or low pH.
(ii)Alteration in the charge state of the E or S or both.

15. (v) Effect of Inhibitors on rate of E catalysed reaction:
Inhibitors are substances that combine with E and decrease its activity.
•Presence of I decreases the rate of E catalysed reaction.
•Inhibitors may be:
i. Irreversible inhibitor
E + I→EI (20)
This inhibitor cannot be removed by dialysis or other means:
Inhibition increases with time.
Examples of irreversible inhibitors;
•CN inhibits xanthine oxidase.
ii. Reversible inhibitors
E + I↔EI (21)

16. The reaction is reversible and the I can be removed by dialysis or other means. These are of
two types:
•Competitive
•Non-competitive
(v)Effect of Activators on rate of E catalysed reactions.
•Some E require activators to increase the rate of reaction.
•Activators cause activation of E-catalysed reaction by either altering the velocity of
the reaction or the equilibrium reached or both.
e.g.:
•Essential activators: Essential for the reaction to proceed. These are recognised as
substrate that is not changed in the reaction e.g. metal ion such as Mg++for kinases
.•Non essential activators: Activator may act to promote a reaction which is capable
of proceeding at a appreciable rate in the absence of activator.

17. Michaelis-menten equation
Step 1. Formation of the ES complex:
E+S
ES
K2
K-1
ES
Step 2.Formation of products:
K-2
K1
E+ P
Assumption 1
E+S
K1
K-1
ES
K2
E+ P
......(1)
......(2)

18. Rate of formation ES = K1 [E] [S]
Rate of brekdown ES = K-1 [ES] + K2 [ES]
= [ES ] [K-
1
+ K2 ]
At equilibrium
K1 [E] [S]= [ES ] [K-1
+ K2 ][E] [S]
[ES]
= [K-1
+ K2 ]
K1
= Km
Total con. Of Enzyme
ET
= [ET] -[ES]E
= [E] + [ES]
......(3)
......(4)
......(5)

19. [ET] -[ES][S]
[ES]
= Km
Km [ES]= ( [ET] -[ES] )[S]
= [ET] [S]-[ES] [S]
Km[ES][ES] [S] + = [ET] [S]
[ES]([S] +Km)= [ET] [S]
[ES] = [ET] [S]
[S] +Km
V0 = K2 [ES]

20. V0 = K2[ET] [S]
[S] +Km
Vmax= K2 [ET]
V0 = Vmax .[S]
[S] +Km Michaelis-menten equation

21. Modification
Vi =
Vmax
.[S][S] +Km
Vi = Vmax
.[S][S] +Km
Km
Vi =Vmax .[S]
Teherefore
Vmax
.[S] [S]
Vi =
(1)
(2)

22. (3)
Vi =
Vmax
.[S][S] +Km
Vmax [S]
2[S]
Vmax [S]
Vi =
Vi =
2

23. Lineweaver-Burk
equation:
[1/v] = [Km(1)/Vmax[S] + (1)/Vmax]

24. REFERENECES
•Fersht A: Structure and Mechanism in Protein Science: A Guide to Enzyme
Catalysis and Protein Folding. Freeman, 1999.
•Schultz AR: Enzyme Kinetics: From Diastase to Multi-enzyme Systems. Cambridge
Univ Press, 1994.
•Segel IH: Enzyme Kinetics. Wiley Inter science, 1975.
•. Smita S. Patel1, Rajiv P. Band war, and Mikhail K. Levin
•Robert Wood Johnson Medical School, Piscataway, New Jersey.
•1Author for correspondence, Tel.: 732-235-3372; Fax: 732-235-4783; E-mail:
patelss@umdnj.edu.Principle of Biochemistry 4th edition, Lehninger