The document discusses factors that affect the rate of enzyme action, including enzyme concentration, substrate concentration, temperature, pH, concentration of coenzymes and activators, time, and inhibitors. It provides details on how each factor influences the reaction rate. Specifically, it explains that enzyme activity is highest when substrate concentration is saturating but not excessive, and when other conditions like temperature and pH are optimal. The document also describes Michaelis-Menten kinetics and how reversible and irreversible inhibition can decrease reaction rates.

1. Factors Affect Rate of
Enzyme Action
DR VISESH KUMAR

2. Factors Affect Rate of Enzyme
Action
 Enzyme concentration
 Substrate concentration
 Temperature
 pH
 Concentration of coenzymes
 Concentration of ion activators
 Time
 Inhibitors

3. Factors affecting the rate of enzyme
action
1- Effect of enzyme concentration
The rate of enzyme action is
directly proportional to the
concentration of enzyme provided
that there are sufficient supply of
substrate & constant conditions.

5. 2- Effect of substrate concentration
-The rate of reaction increases as the
substrate concentration increases up to
certain point at which the reaction rate is
maximal (Vmax.)
At Vmax, the enzyme is completely
saturated with the substrate any increase
in substrate concentration doesn't affect
the reaction rate.

7. A plot of reaction velocity versus substrate
concentration
Here varying amounts of
substrate are added to a
fixed amount of enzyme. The
reaction velocity is measured
for each substrate
concentration and plotted.
The resulting curve takes the
form of a hyperbola (a
mathematical function in
which the values initially
increase steeply but
eventually approach a
maximum level).

8. Measured at the very beginning of a reaction when very little P has been
made
Enzyme present in nanomolar quantities, whereas [S] may be five or six
orders of magnitude higher

9. At relatively low concentrations of substrate, V0 increases almost linearly
with an increase in [S]
At higher substrate concentrations,V0 increases by smaller and smaller
amounts in response to increases in [S]
A point is reached beyond which increases in V0 are vanishingly small as
[S] increases
This plateau-like V0 region is close to the maximum velocity, Vmax.
The kinetic pattern led Victor Henri, to propose in 1903 that the
combination of an enzyme with its substrate molecule to form an ES
complex is a necessary step in enzymatic catalysis
This idea was expanded into a general theory of enzyme action,
particularly by Leonor Michaelis and Maud Menten in 1913.

10. They postulated that the enzyme first combines reversibly with its substrate to
form an enzyme-substrate complex in a relatively fast reversible step
The ES complex then breaks down in a slower second step to yield the free
enzyme and the reaction product P:
Because the slower second reaction must limit the rate of the overall reaction,
the overall rate must be proportional to the concentration of the species that
reacts in the second step, that is, ES
the pre–steady state, during which the concentration of ES builds up
The reaction quickly achieves a steady state in which [ES] remains
approximately constant over time.
The concept of a steady state was introduced by G. E. Briggs and Haldane in
1925

11. The more ES present, the faster ES
will dissociate into E + P or E + S.
Therefore, when the reaction is
started by mixing enzymes and
substrates, the [ES] builds up at
first, but quickly reaches a
STEADY STATE, in which [ES]
remains constant. This steady state
will persist until almost all of the
substrate has been consumed.

12. • Michaelis Menten equation: describe the rate of steady
state enzymatic reactions with relation to
the concentration of a substrate
Where, S = substrate
E = enzyme
ES = enzyme-substrate concentration
P = product
k1, k-1 and k2 are rate constants

13. – Where, vo = initial reaction velocity
Vmax = maximal velocity
Km = Michaelis constant = (k2 + k-1 )/ k1
[S] = substrate concentration
Criteria for Michaelis kinetics:
• Steady state reaction: rate of formation of [ES] equal rate of
breakdown of [ES] i.e. concentration of [ES] does not change
• Concentration of substrate is much greater than the
concentration of enzyme
• Initial velocity, vo measured as soon as enzyme and substrate
are mixed i.e. vo is dependent on [S] and Km.

14. Michaelis –Menten Kinetic theory
• M & M model accounts for the kinetic properties of some
enzymes. It helps to describe many enzymatic reactions under
the following assumptions:
 The reaction has only one substrate
 The substrate concentration is much higher than that of the
enzyme
 Only the initial velocity is measured.

15. Michaelis constant (Km)
- It is the substrate concentration
that produces half maximum
velocity of enzyme

16. vo dependent on [S] and Km
1. When [S] <<< Km, Km + [S] equivalent to Km,
vo = Vmax [S]
Km
since Vmax and Km are constant, vo α [S]
2. When [S] >>> Km, Km + [S] equivalent to [S]
vo = Vmax
3. When [S] = Km,
vo = Vmax/ 2

17. Importance of Michaelis Menten Kinetics
1. Characteristics of Km:
– Km is characteristic of an enzyme and its particular substrate,
reflects the enzyme affinity
– Numerically Km equal to substrate concentration at which
reaction velocity is equal to ½ Vmax.
– Km does not vary with enzyme concentration
– Small Km reflects high affinity between enzyme and substrate
– High Km reflects low affinity between enzyme and substrate

18. Effect of substrate concentration on reaction velocities for two
enzymes: enzyme 1 with a small Km, and enzyme 2 with a large Km

19. Lineweaver Burk Plot
• In Michaelis Menten equation, when vo is plotted against
[S], as the curve is hyperbolic it is hard to calculate Vmax.
• If equation is reciprocated and the graph is plotted, a
straight line is obtained. This plot is called Lineweaver Burk
plot or Double reciprocal plot.
– Can be used for calculating Km and Vmax, as well as to
determine the mechanism of action of enzyme inhibitors.

20. Measuring Km and V max
• Curve-fitting algorithms can
be used to determine Km and
Vmax from v vs. [S] plots
• Michaelis-Menton equation
can be rearranged to the
“double reciprocal” plot and
Km and Vmax can be
graphically determined

21. Temperature
The effect of temperature on reaction rate is due
to:
1- Increase of temperature increase the initial
energy of substrate and thus decrease the
activation energy
2- Increase of collision of molecules: more
molecules become in the bond forming or bond
breaking distance.

22. After the optimum temperature,
the rate of reaction decrease due
to denaturation of the enzyme (60-
65 C).

23. 4- Effect of PH
- Each enzyme has an optimum PH at
which
its activity is maximal
• E.g. Optimum PH of pepsin = 1.5 - 2
• Optimum PH of pancreatic lipase = 7.5 -
8
• Optimum PH of salivary amylase = 6.8

24. Change of PH above or below optimum PH
decrease rate of enzyme action due to:
1- The enzyme activity depends on the
ionization state of both enzyme and
substrate which is affected by PH.
2- Marked change in PH will cause
denaturation of enzyme.

25. 5- Concentration of coenzymes:
In the conjugated enzymes that need
coenzymes, the increase in the coenzyme
concentration will increase the reaction
rate

26. 6- Concentration of ion activators:
The increase in metal ion activator
increase the reaction rate
Enzymes are activated by ions:
1- Chloride ion activate salivary amylase
2- Calcium ion activate thromobokinase
enzyme

27. 7- Effect of time:
• In an enzymatic reaction, the rate of
reaction is decreased by time.
• This is due to:
1- The decrease in substrate concentration.
2- The accumulation of the end products.
3- The change in PH than optimum PH.

28. 8- Presence of enzymes inhibitor:
Presence of enzyme inhibitor
decreases or stops the
enzyme activity.

29. Enzyme Inhibiton
Any substance that can diminish the velocity
of an enzyme catalyzed
These include drugs, antibiotics, poisons,
and anti-metabolites.
Useful in understanding the sequence of
enzyme catalyzed reactions, metabolic
regulation, studying the mechanism of cell
toxicity produced by toxicants.
Forms the basis of drug designing.

30. Types of Enzyme Inhibiton
• Reversible inhibitors
• Irreversible inhibitors

31. Reversible inhibitors can be
classified into :
• Competitive
• Non-competitive
• Un-competitive

32. Classes of Inhibition
Two real, one hypothetical
• Competitive inhibition - inhibitor (I) binds only
to E, not to ES
• Noncompetitive inhibition - inhibitor (I) binds
either to E and/or to ES
• Uncompetitive inhibition - inhibitor (I) binds
only to ES, not to E. This is a hypothetical case
that has never been documented for a real
enzyme, but which makes a useful contrast to
competitive inhibition

33. • Competitive inhibitor:
– Inhibitor binds reversibly at same site of substrate;
compete with substrate
– Inhibition is reversed by increasing [S]
– Inhibitor increases the apparent Km, but Vmax is
not affected
– E.g.:
• Statin drugs inhibit HMG CoA Reductase
• Inhibitor malonate competes with succinate for
succinate dehydrogenase

34. Competitive Inhibition

35. A. Effect of a competitive inhibitor
on the reaction velocity (vo)
versus substrate ([S]) plot.
B. Lineweaver-Burk plot of
competitive inhibition of
an enzyme.

36. Lovastatin competes with
HMG-CoA for the active site
of HMG-CoA reductase.

37. Succinate dehydrogenase
Reaction
Malonate is a
competitive inhibitor

38. A competitive inhibitor

39. • Non competitive inhibition:
– Inhibitor and substrate binds at different sites
– Inhibitor binds either free enzyme or [ES] complex
– Vmax decreases as inhibition cannot be overcome by
increasing [S]
– Km is not affected as inhibitor does not interfere
substrate binding to enzyme
– Eg.:
• Ferrochelatase catalyses Fe2+ insertion into
protoporphyrin during Heme synthesis
–Lead is the non competitive inhibitor

40. Non-Competitive Inhibition

41. A. Effect of a noncompetitive
inhibitor on the reaction velocity
(vo) versus substrate ([S]) plot.
B. Lineweaver-Burk plot of
noncompetitive inhibition of an
enzyme.

42. Un-competitive Inhibiton
Binds only to the enzyme-substrate
complex.
Does not have the capacity to bind to the
free enzyme.
Not overcome by increasing substrate
concentration.
Both the Km and Vmax are reduced.

43. Uncompetitive inhibition
• Uncompetitive inhibitors
bind only the ES complex,
not free enzyme (E).
• Both Vmax and Km are
affected by inhibitor, [I].
• Km is affected because the
ESI complex prevents E and S
from dissociating.
• Vmax is affected because the
ESI complex can’t form
product (ES is trapped as ESI
at saturating substrate
concentration).

44. Un-competitive Inhibiton
Enzyme
ES Complex
+ Inhibitor
ESI complex

45. Irreversible inhibition
• Inhibitor act irreversibly by chemically modifying the
enzyme
• involve formation of covalent bonds between inhibitor
and aminoacyl residues on enzymes,
• Covalent changes are relatively stable; and enzyme
remains inactive even after removal of inhibitor from
surrounding medium
• E.g.: Heavy metal, etc.

46. Irreversible inhibition
• Inhibition of Cyclooxygenase (COX)by aspirin.
• Iodoacetate, heavy metal ions, oxidising
agents form covalent bonds with functional
group of enzymes.
• Suicide Inhibition is a type of irreversible
inhibition.

47. Mechanism based/ Suicide inhibition
• Suicide inhibitors, specialized substrate analogs, that
are transformed into a more highly reactive group by
the target enzyme
• Forms covalent bond and blocks the function of a
catalytically active residue leading to irreversible
inactivation of the enzyme.
• Basis for development of enzyme specific drugs
• Eg:
– Allopurinol used for gout/ hyperuricemia
– Competes with hypoxanthine for xanthine oxidase
– Allopurinol converted into more potent inhibitor,
alloxanthine

48. Irreversible Inhibitor: Allopurinol