Factors effecting enzyme activity
Objective
At the end of this lecture,
student will be able to
• Explain the factors affecting enzyme activity
• Describe enzyme kinetics
• Outline Michealis-Menton Equation
Factors
effecting enzyme activity
• The important factors that influence the velocity of the
enzyme reaction are
1. Concentration of substrate
2. Concentration of enzyme
3. Temperature
4. PH
5. Effect of product concentration
6. Effect of activators
7. Effect of time
8. Effect of light and radiation
1.
Concentration of substrate
• Increase in the substrate concentration gradually
increases the velocity of enzyme reaction within the limited range of substrate
levels
• A rectangular hyperbola curve is obtained when velocity is
plotted against the substrate concentration
• 3 distinct phases of the reaction are observed in the
graph;
A-linear; B-curve; C-almost unchanged
Michelis-Menten
Equation:
Order of reaction:
• When the velocity of the reaction is proportional to the
substrate concentration (i.e [S] ˂ KM), the rate of the reaction is said to be
first order with respect to substrate
• When the [S] ˃ KM, the rate of reaction is independent of
substrate concentration and the reaction is said to be zero order
• Enzyme kinetics and K value: The enzyme (E) and substrate
(S) combine with each other to form an unstable enzyme-substrate complex (ES)
and product (P)
• Here kl, k2 & k3 represent the velocity constants for the
respective reactions as indicated by arrows
• KM Michaelis-Menten constant (or Brigrs and Haldane’s constant)
is given by the formula
• The following equation is obtained after suitable algebraic
manipulation
• Let us assume that the measured velocity (v) is equal to ½
Vmax. Then the equation (1) may be substituted as follows;
• K stands for a constant and m stands for Michaelis (in KM)
• KM or Michaelis-Menten constant is defined as the substrate
concentration (expressed in moles/l) to produce half-maximum velocity in an
enzyme catalysed reaction.
• KM value is a constant and a characteristic feature of a
given enzyme. lt is representative for measuring the strength of ES complex
• A low KM value indicates a strong affinity between enzyme
and substrate, whereas a high KM value reflects a weak affinity between them
• For majority of enzymes, the KM values are in the range of
10-5 to 10-2 moles
Line weaver-Burk
double reciprocal plot:
• For the determination of KM value, the substrate
saturation curve is not very accurate, Since Vmax is approached asymptotically
• By taking the reciprocals of the equation (1) a straight
line graphical representation is obtained
• The above equation is similar to y = ax + b
• Therefore, a plot of the reciprocal of the velocity [1/v]
VS reciprocal of the substrate concentration [1/[s]] gives a straight line
• Here the slope is KM/Vmax and whose y intercept is 1/Vmax
• By this plot, it is much easier to calculate the KM from
the intercept on x-axis which is -(1/Km)
• Further, the double reciprocal plot is useful in understanding
the effect of various inhibitions
2.
Concentration of enzyme
• Increase in concentration of the enzyme, increased the
velocity of the reaction
• This property of enzyme is used to determining the serum
enzymes for the diagnosis of diseases
• By using a known volume of serum and keeping all the other
factors (substrate, pH, temperature etc.) at the optimum level, the enzyme could
be assayed in the laboratory
3.
Temperature:
• Velocity of an enzyme reaction increases with increase in
temperature up to a maximum and then declines
• A bell-shaped curve is usually observed
• Temperature coefficient or Q10 is defined as increase in
enzyme velocity when the temperature is increased by 10oC
• For a majority of enzymes, Q10 is between 0oC
and 40oC
• The optimum temperature for most of the enzymes is between
40oC- 45oC
• Few enzymes (e.g. venom phosphokinases) are active even at
100oC
• Some plant enzymes like urease have optimum activity
around 60oC
• In general, when the enzymes are exposed to a temperature
above 50oC Denaturation occurs and the enzymes become inactive
4. pH
• Increase in the hydrogen ion concentration (pH)
considerably influences the enzyme activity and a bell-shaped curve is normally
obtained
• Each enzyme has an optimum pH at which the velocity is
maximum, Below and above this pH, the enzyme becomes totally inactive
• Most of the enzymes show optimum activity around neutral
pH (6-8)
• However some enzymes like pepsin at (1-2), acid
phosphatase (4-5) and alkaline phosphatase (10-11) are active
5. Effect
of product concentration
• The accumulation of reaction products generally decreases
the enzyme velocity
• Here, the products combine with the active site of enzyme
and form a loose complex and thus inhibit the enzyme activity
• But in the living system, this type of inhibition is
generally prevented by a quick removal of products formed
6. Effect
of activators
• Some of the enzymes require certain inorganic metallic
cations like Mg2+, Mn2+,zn2+, ca2+, co2+, cu2+, Na+, K+ etc for their optimum
aciivity
• Rarely, anions are also needed for enzyme activity e.g.
chloride ion amylase
• Metals function as activators of enzyme velocity through
various mechanisms
Combining with the substrate
Formation of ES-metal complex
Direct participation in the reaction
Bringing a conformational change in the enzyme
• Two categories of enzymes requiring metals for their
activity are distinguished
Metal activated
enzymes: here metal is not tightly held by the enzyme and can be exchanged
easily with other ions e.g. ATPase (Mg2+ and Ca2+) Enolase (Mg2+)
Metalloenzymes:
These enzymes hold the metals rather tightly which are not readily exchanged. e.g.
alcohol dehydrogenase, carbonic anhydrase, alkaline phosphatase,
carboxypeptidase and aldolase contain zinc
Phenol oxidase (copper), Pyruvate oxidase
(manganese), Xanthine oxidse (molybdenum), Cytochrome oxidase (Iron and copper)
7. Effect
of time:
• Under ideal and optimal conditions, the time required for
an enzyme reaction is less. Variations in the time of the reaction are
generally related to the alterations in pH and temperature
8. Effect
of light and radiation
• Exposure of enzymes to ultraviolet, beta, gamma and X-rays
inactivates certain enzymes due to the formation of peroxides. E.g. UV rays inhibit
salivary amylase activity
Summary
• Increase in the substrate concentration gradually
increases the velocity of enzyme reaction within the limited range of substrate
levels
• Increase in concentration of the enzyme, increased the
velocity of the reaction
• Velocity of an enzyme reaction increases with increase in temperature
and pH up to a maximum and then declines
• The accumulation of reaction products generally decreases
the enzyme velocity
• Exposure of enzymes to ultraviolet, beta, gamma and X-rays
inactivates certain enzymes