For MD, MBBS, BDS & Nursing

1. Factors affecting enzyme
action
Prof. Dr. Viyatprajna Acharya

2. Velocity/ rate of enzyme
reactions
 Rate of change of substrate to product per
unit time
Ex- μmol product/ min
Equilibrium constant of the reaction
Keq = K forward reaction
K backward reaction
A+ B C+ D

3.  At equilibrium- forward & backward
reaction rates are equal
 Equilibrium is a dynamic state
 Numerical value of Keq can be calculated
by knowing the concentration of
substrates and products
 Keq > 1 – rightward reaction is favored
 Conc. Of enzyme doesn’t affect Keq

4. Enzymes can be isolated and properties
can be studied in vitro
Factors affecting enzymes are:
1. Concentration of enzyme
2. Concentration of substrate
3. Concentration of product
4. Temperature
5. pH
6. Activators
7. Time
8. Light & radiation
9. Inhibitors

5. 1. Enzyme concentration
affecting enzyme activity
 When substrate is sufficient,
Rate of reaction is proportional to
Enzyme concentration
 Unit of enzyme activity- IU, Katal
(Kat), U, KAU

7. 2. Substrate concentration
affecting enzyme activity
E + S ↔ ES ↔ E + P
A
B
C
3 Phases
A. At low substrate
conc.– V α [S]
B. [S] not directly
proportional to V
C. Reaction
independent of [S]

8. Most of the enzymes follow Michelis-
Menten kinetics

9. MICHELIS-MENTEN EQUATION
Enzyme combines reversibly with substrate to form ES.
Breakdown of ES to product is irreversible.
E= Enzyme
S= Substrate
P= Product
Es= Enz- substrate complex
K1, K-1, K2= Rate constants

10. Michelis constant
Km = K1+ K2
K
1
After algebraic manipulation

11. If V= Vmax
2
Substituting,
V0 = Vmax · [S] / Km + [S]
Vmax/2 = Vmax · [S] / Km + [S]
2[S] = Km + [S]
Km = 2[S] – [S] = [S]
Km is equivalent to the substrate
concentration at which
V0 is one-half Vmax.

12.  At high [S], [S] >>> Km ,
therefore V0 = Vmax
 At low [S], [S] <<< Km ,
therefore V0 = Vmax· [S] / Km

13. Km/ Michelis constant
 It’s the substrate concentration (expressed in
moles/lit) at half-maximal velocity.
 50% of enzyme molecules are bound with
substrate molecules at that particular substrate
concentration
 Km is independent of enzyme concentration
 Expressed in moles/lit
 Km is a constant for an enzyme. It’s the
characteristic feature of a particular enzyme for a
specific substrate- Signature of the enzyme

14.  Km is the representative of measuring the
strength of the ES complex
Low Km – strong affinity between enzyme
and substrate
Ex: Glucokinase– Km= 10 mmol /lit
Hexokinase– Km= 0.05 mmol/lit
So what’s the inference??

15. 50% molecules of Hexokinase are
saturated even at a lower conc. Of
glucose.
 When [S] << Km → reaction is first-
order
 When [S] >> Km → reaction is zero-
order

16. Double reciprocal (Lineweaver -Burk plot)
Purpose:
 To calculate Km
 When impracticably very high conc. Of substrate
required to achieve Vmax
 Gives a straight line; easy to calculate
 Gives idea of different inhibitions

18. Co-operative binding
 Some enzymes don’t follow M-M equation
strictly
 Enzymes having many subunits
 Binding of substrate on one subunit enhances
binding with other subunits
 Follow Hill equation

20. Dixon Plot
 [S] is kept constant and Velocity (V) is
measured at different concentrations
of Inhibitors (I)
 Used for determining inhibition
constants
 Plot against 1/V Vs [I] is done
 A straight line obtained

21. Dixon plot for non-
competitive inhibition

22. Take a break!!

23. 3. Effect of concentration of
products
↑ Product concentration → ↓ reaction rate
4. Effect of Temperature
↑ temperature
↓
↑ molecular collision
↓
↑ interaction
↓
Faster reaction

24.  Optimum temperature for most enzymes– 300c-400c
 Most of human enzymes- 370-380c
 Exception- Venom phosphokinase, muscle adenylate
kinase– 1000c
 Urease– 600c
 Taq Polymerase- used in PCR; sustains 1000c

25. Temperature coefficient (Q10)
Increase in enzyme velocity when the
temperature is increased by 100c
 Usually rate of reaction doubles for
most enzymes when temp is raised by
100c- but this happens till 500c

26. 5. Effect of pH
pH change alters:
 Ionization states of the amino acid residues
present in the active site
 Ionization state of substrate
 May dissociate apoenzyme from cofactor
 Drastic change denatures the enzyme protein
Optimum pH is different for different enzymes
Mostly 6-8
Exception: Pepsin– 1-2
ALP– 9-10
Acid phosphatase – 4-5

27. pH affecting enzyme activity
A bell-shaped curve

28. 6. Effect of activators
 Inorganic metal ions increase enzyme
activity
Ex: Mg, Mn, Zn, Co, Cu etc
Cl - - Salivary amylase
Ca++ - Lipase

29. 7. Effect of Time
 Under ideal and optimal conditions,
the time required for an enzyme
reaction is less

30. 8. Effect of light and
radiation
 UV rays,β, γ, X- rays inactivate

31. Forgive your enemies, but do not forget their
names
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