Here is just a quick recap about factors affecting enzymes activity.
WHAT ARE FACTORS THAT AFFECT ENZYMES ACTIVITY?
– Substrate concentration
– Enzyme concentration
HOW DOES SUBSTRATE CONCENTRATION AFFECTS ENZYMES ACTIVITY?
As substrate concentration increases the rate at which enzyme substrate complex formed increases because there are more substrate available for free enzymes active site to bind with. Also with an increase in substrate concentration the greater the collision frequency of substrate to enzyme active site. However at Vmax all the enzyme active site is occupied by substrate therefore the rate of reaction is constant so increasing [S] doesn’t affect the rate of reaction. When observing the effects of substrate concentration on enzyme activity the following variables must be kept constant pH, temperature and enzyme concentration. The effect of substrate concentration can be illustrated by using either line weaver Burk plot (equation) or Michaelis–Menten graph (equation).
FIGURE 1: ILLUSTRATES THE EFFECT OF SUBSTRATE CONCENTRATION ON ENZYME ACTIVITY.
HOW DOES pH AFFECT ENZYME ACTIVITY?
When the pH is too acidic or too basic for an enzyme, its hydrogen bonds begin to break resulting in the enzyme active site losing its shape. Each enzyme operates within an pH range (optimal pH). For example: lipase in the stomach operates within the optimum pH 4-5.
FIGURE 2: ILLUSTRATES THE EFFECT OF pH ON ENZYME ACTIVITY.
HOW DOES TEMPERATURE AFFECT ENZYME ACTIVITY?
As temperature increase the rate of reaction increases however beyond the point of optimum temperature the rate of enzyme catalyzed reaction decreases because the hydrogen bond and the hydrophobic interaction in the enzyme structure is being broken. When this occur enzyme losses it shape and the substrate is unable to bind properly to the active site thus reducing enzyme activity. Furthermore, as temperature increases the amount of kinetic energy increases in the system thus increasing the rate of reaction since the collision frequency between the substrate and the enzyme active site increases. Also as temperature increases the substrate molecules gain more energy to overcome the activation barrier hence forming more ES complex which alternately increases rate of enzyme reaction.
FIGURE 2: ILLUSTRATES THE EFFECT OF TEMPERATURE ON ENZYME ACTIVITY.
HOW DOES INHIBITORS AFFECT ENZYME ACTIVITY?
There are irreversible inhibitors and reversible inhibitors which affect rate of enzyme reaction. Reversible inhibitors binds to the enzyme by non- covalent bond therefore dilution of the enzyme-inhibitor complex releases the inhibitor and the enzyme can carry on its activity. There are four types of reversible inhibitors are competitive, uncompetitive, mixed and non-competitive.
1. Competitive inhibitors compete with the substrate for the active site, therefore the shape of the inhibitor resemble the shape of the substrate. When the inhibitor binds to the active site it reduces the rate of enzyme reaction since the substrates unable to bind to the active site because the inhibitor is currently occupying the active site . Competitive inhibitor increases Km therefore more substrate is needed to achieve ½ Vmax. Vmax however is unaffected by competitive inhibitor.
FIGURE 3: ILLUSTRATES THE EFFECT OF COMPETITIVE INHIBITOR ON Vmax AND Km IN LINEWEAVER- BURK PLOT.
2. Non-competitive inhibitor binds to free enzyme or ES complex; it doesn’t bind to the active site. Therefore the shape of the inhibitor is different from the substrate. This type of inhibitor reduces the ability of the enzyme to convert substrate into product. Vmax is reduced and Km remains the same since the substrate can still bind to the active site as well as before the inhibitor is present.
FIGURE 4: ILLUSTRATES THE EFFECT OF NON-COMPETITIVE INHIBITOR ON Vmax AND Km IN MICHAELLIS MENTEN CURVE AND LINEWEAVER- BURK PLOT RESPECTIVELY.
3. Uncompetitive inhibitor occurs when the inhibitor binds only to the ES complex. They do not bind to free enzymes. It reduces both Vmax and Km in the same amount. The slope of the graph will remain constant because both the Km and Vmax will be changing proportionally to each other and so the line will simply be observed to shift up and to the left.
FIGURE 5: ILLUSTRATES THE EFFECT OF UNCOMPETITIVE INHIBITOR ON Km AND Vmax IN A LINEWEAVER BURK PLOT.
4. Mixed inhibitor binds to free enzyme or enzyme substrate complex. They do not bind to the active site. When the inhibitor binds to the enzymes it changes the shape of the enzyme thereby reducing the affinity of the substrate to the enzyme active site. Vmax is always reduced whereas Km is either increase or decrease.
Mixed inhibitor may seem similar to non- competitive inhibitor however they are different. When non-competitive inhibitors binds to the enzymes it form the EIS complex that caused the product not to be formed whereas in mixed inhibitor when the inbitor bind to the enzyme forming the EIS complex some product are still formed although the inhibitor is present.
FIGURE 6: ILLUSTRATES THE EFFECT OF MIXED INHIBITOR ON Km AND Vmax USING A LINEWEAVER BURK PLOT.
Cox. M.M. and David L. Nelson 1984. Lehninger; Principles of Biochemistry. Fourth Edition.
Worthington Biochemical Corporation.2013. “Introduction to enzymes.” http://www.worthington-biochem.com/introBiochem/substrateConc.html
mymcat.com.2010. “Enzyme Inhibitor.” Accessed March 7th 2013. http://www.mymcat.com/wiki/Enzyme_Inhibition#Noncompetitive