Factor affecting enzyme action

FACTORS AFFECTING ENZYME

ACTION
 MEDICAL AND BIOLOGICAL
IMPORTANCE
1. all enzymatic reactions must proceed at appropriate
rates. Alterations may disturb tissue homeostasis
2. Any alteration in intracellular pH disturbs rates of
enzyme reactions
3. Organs for transplantation, blood and serum are
preserved at low temperature as soon as they are
removed from body because enzymatic reactions
proceed at much lower rate at low temperature. Under
such conditions, O2 demand of cells decreases, so cells of
the organs or fluids survive with available O 2 for
sometime.
4. In fever and hypothermia - temperature influences rate
of enzyme reaction
5. An understanding of factors affecting enzyme action is
required for development of drugs. Many drugs act by
decreasing rate of key metabolic reaction by blocking
that particular enzyme.
e.g.lovastatin is used in treatment of atherosclerosis is an
inhibitor of HMGCoA reductase, a cholesterol producing
enzyme.
6. Some poisons work by abolishing (affecting) essential
enzymatic reactions
1. Enzyme concentration
2. Temperature
3. Hydrogen ion concentration or pH
4. Substrate concentration
5. Inhibitors
6. Product concentration
7. Activators
8. Physical agents
 Enzyme concentration
 The rate of enzyme catalyzed reaction is directly proportional to
the concentration of enzyme.

 The plot of rate of catalysis versus enzyme concentrations a

straight line
 Temperature
 Increase with temperature
 Bell shape curve
 Q10 (temperature coefficient)- factor by which the
rate of biological reaction increases for a 10ºC
increase in temperature
 Optimum temperature
 Mostly at body temperature
 Some enzyme may be active above body
temperature e.g. sanke venom phosphokinase,
muscle adenylate kinase, urease, enzymes in
thermophillic bacteria
• Rise or fall in enzyme activity with
temperature is prominent survival feature in
“Cold blooded” animals
• In mammals- assumes physiological
importance e.g. fever, hypothermia
 pH
• Bell shape curve
• Optimum pH
• Most show at neutral pH (6-8)
Since enzymes are proteins pH changes affect.
1. Charged state of catalytic site
2. Conformation of enzyme molecules
 Optimum pH for various enzyme
• Trypsin- 7.6
• Pepsin- 2-2.5
• Acid phosphatase- 5
• Alkaline phosphatase- 9-10
• Enzymes from fungi- 4-6
 Product concentration
• Accumulation decreases the velocity
• In biological system this is prevented by quick
removal product
 Activators
• Inorganic metallic cation/anions acts as
activators by combining with substrate, ES
complex, change in conformation of active
site
• Metal activated enzymes- e.g. ATPase,
Enolase
• Metalloenzyme- e.g.Pyruvate oxidase,
cytochrome oxidase
 Inhibitors
• Make active site unavailable to substrate or
• Change enzyme structrure
 Physical agents
• Light, radiation ( u.v., X- rays, gamma rays etc)
e.g. salivary amylase- activity increased by red/
blue light whereas decreased by u.v. light
 Substrate concentration
• Rectangular hyperbola (Michaelis plot)
• Initial velocity- velocity when little substrate is
reacted
Reasons for the three phases of the curve can be
interpreted
1. In the first phase, substrate concentration is low and
most of the enzyme molecules are free so they combine
with the substrate molecules. Therefore, velocity is
proportional to substrate concentration. At this state,
enzymatic reaction shows first-order kinetics

2. In the second phase, half of the enzyme molecules are

bound to substrate, so the velocity is not proportional to
substrate concentration. At this stage, enzymatic
reaction shows mixed-order kinetics

3. In the third phase, all the enzyme molecules are bound

to substrate, so velocity remain unchanged because free
enzyme is not available though the substrate is in excess.
At this stage enzymatic reaction shows zero-order
kinetics
A. Low [S] B. 50% [S] or Km C. High, saturating [S]
 Steady State Assumption
 The M-M equation was derived in part by making several
assumptions. An important one was: the concentration of
substrate must be much greater than the enzyme
concentration.
 In the situation where [S] >> [E] and at initial velocity rates, it
is assumed that the changes in the concentration of the
intermediate ES complex are very small over time (vo).
 This condition is termed a steady-state rate, and is referred
to as steady-state kinetics. Therefore, it follows that the
rate of ES formation will be equal to the rate ES breakdown.
Michaelis-Menten Equation Derivation

2 4
Rate of ES formation = k1([ET] - [ES])[S] (where
[ET] is total concentration of enzyme E and k 4 is
considered neglible)
Rate of ES breakdown to product = k 2[ES] +
k3[ES]
• Thus for the steady state assumption:

• k1([ET] - [ES])[S] = k3[ES] + k2[ES]

• This equation is the basis for the final

Michaelis-Menten following algebraic
rearrangement and substitution of Km
and Vmax terms
 Michaelis-Menten Equation

vmax S 
v
K M  S 

In which:
v initial reaction velocity at [S]
KM the Michaelis constant
vmax the maximum possible initial reaction velocity
 Michaelis Constant (Km )
The substrate concentration that produces half
the maximal velocity (Vmax/2) is known as
Michaelis constant (Km )
 Meaninig of Km
Michaelis constants have been determined for many of the
commonly used enzymes. The size of Km tells us several
things about a particular enzyme:

1. A small Km indicates that the enzyme requires only a

small amount of substrate to become saturated. Hence,
the maximum velocity is reached at relatively low
substrate concentrations.

2. A large Km indicates the need for high substrate

concentrations to achieve maximum reaction velocity.

 The substrate with the lowest Km upon which the

enzyme acts as a catalyst is frequently assumed to be
enzyme's natural substrate, though this is not true for all
enzymes.

 A Km of 10-7 M indicates that the substrate has a greater

affinity for the enzyme than if the K is 10-5 M.
 Significance of Km
1. enzyme kinetic constant.
2. Indicates the substrate concentration required for the
enzyme to work efficiently
3. Low Km indicates high affinity of enzyme towards substrate.
And vice-versa. Hence,(Km α 1/affinity)
e.g. Hexokinase and glucokinase
Km of hexokinase is low (1 × 10–5 M) whereas Km of glucokinase
is high (2.0 × 10–2 M)

4. Km is required when enzymes are used as drugs

5. Use of enzymes in immunodiagnostics (ELISA) require Km of
the enzyme
 The Catalytic Constant kcat

 At high substrate concentration the overall velocity of the

reaction is Vmax and the rate is determined by the enzyme
concentration.
 The rate constant observed under these conditions is called the
catalytic constant, kcat, defined as:

 kcat indicates the maximum number of substrate molecules

converted to product each second by each active site. This is
called turnover number.
 The catalytic constant measures how fast a given enzyme can
catalyze a specific reaction (describing the effectiveness of an
enzyme)

 The unit for kcat is s-1 (for the most enzymes, kcat is 102 to 103 s-1)
 Lineweaver-BurK Plot

V = reaction velocity (the reaction rate),

Km = Michaelis-Menten constant,
Vmax = maximum reaction velocity
[S] = the substrate concentration