ENZYMOLOGY

DR. BUSHRA MAHMOOD HUSSEIN

CHEMICAL PATHOLOGIST
FACTORS AFFECTING ENZYME ACTIVITY

1.Substrate Concentration
• The rate at which an enzymatic reaction proceeds and whether the forward or reverse
reaction occurs depend on several reaction conditions. One major influence on enzymatic
reactions is substrate concentration.
• In 1913, Michaelis and Menten hypothesized the role of substrate concentration in the
formation of the enzyme–substrate (ES) complex.
• According to their hypothesis, represented in Figure 1-1, the substrate readily binds to free
enzyme at a low substrate concentration.
• With the amount of enzyme exceeding the amount of substrate, the reaction rate steadily
increases as more substrate is added.
• The reaction is following first-order kinetics because the reaction rate is directly proportional
to substrate concentration.
• Eventually, however, the substrate concentration is high enough to saturate all available
enzyme, and the reaction velocity reaches its maximum.
• When the product is formed, the resultant free enzyme immediately combines with excess
free substrate. The reaction is in zero-order kinetics, and the reaction rate depends only on
enzyme concentration.
FIGURE 1-1 MICHAELIS-MENTEN CURVE OF VELOCITY VS. SUBSTRATE
CONCENTRATION FOR ENZYMATIC REACTION. KM IS THE SUBSTRATE
CONCENTRATION AT WHICH THE REACTION VELOCITY IS HALF OF THE
MAXIMUM LEVEL.
• The Michaelis- Menten hypothesis of the relationship between reaction velocity and substrate
concentration can be represented mathematically as follows:
• V= V max [S]
Km+[S]
• Where V is the measured velocity of reaction, V max is the maximum velocity, [S] is the
substrate concentration, and Km is the Michaelis-Menten constant of enzyme for a specific
substrate.
FACTORS AFFECTING ENZYME ACTIVITY

2.Enzyme Concentration
• Because enzymes catalyze physiologic reactions, the enzyme concentration affects
the rate of the catalyzed reaction.
• As long as the substrate concentration exceeds the enzyme concentration, the
velocity of the reaction is proportional to the enzyme concentration. The higher the
enzyme level, the faster the reaction will proceed because more enzyme is present to
bind with the substrate.
FACTORS AFFECTING ENZYME ACTIVITY

3.pH
• Enzymes are proteins that carry net molecular charges. Changes in pH may denature
an enzyme or influence its ionic state, resulting in structural changes or a change in
the charge of an amino acid residue in the active site.
• Hence, each enzyme operates within a specific pH range and maximally at a specific
pH. Most physiologic enzymatic reactions occur in the pH range of 7.0 to 8.0, but
some enzymes are active in wider pH ranges than others.
 FACTORS AFFECTING ENZYME ACTIVITY
4.Temperature
• Increasing the temperature usually increases the rate of a chemical reaction by increasing
the movement of molecules, the rate at which intermolecular collisions occur, and the
energy available for the reaction.
• This is the case with enzymatic reactions until the temperature is high enough to denature
the protein composition of the enzyme.
• For each 10° increase in temperature, the rate of the reaction will approximately double
until, of course, the protein is denatured.
• The rate of denaturation increases as the temperature increases and is usually significant
at 40°C to 50°C.
 FACTORS AFFECTING ENZYME ACTIVITY
5.Cofactors
• Cofactors are nonprotein entities that must bind to particular enzymes before a reaction
occurs. Common activators (inorganic cofactors) are metallic (Ca2+, Fe2+, Mg2+, Mn2+,
Zn2+, and K+) and nonmetallic (Br- and Cl–).
• The activator may be essential for the reaction or may only enhance the reaction rate in
proportion with concentration to the point at which the excess activator begins to inhibit the
reaction.
• Some common coenzymes (organic cofactors) are nucleotide phosphates and vitamins.
Coenzymes serve as second substrates for enzymatic reactions. When bound tightly to the
enzyme, coenzymes are called prosthetic groups.
EFFECTS OF INHIBITORS ON ENZYME ACTIVITY

• Enzyme inhibitors are substances which alter the catalytic action of the enzyme and
consequently slow down, or in some cases, stop catalysis. There are three common types of
enzyme inhibition - competitive, non-competitive and substrate inhibition.
• Most theories concerning inhibition mechanisms are based on the existence of the enzyme-
substrate complex ES.
• Competitive inhibition occurs when the substrate and a substance resembling the substrate
are both added to the enzyme. A theory called the "lock-key theory" of enzyme catalysts can
be used to explain why inhibition occurs.
• The lock and key theory utilizes the concept of an "active site." The concept holds that one
particular portion of the enzyme surface has a strong affinity for the substrate.
• The substrate is held in such a way that its conversion to the reaction products is more
favorable. If we consider the enzyme as the lock and the substrate the key (Figure 9) - the
key is inserted in the lock, is turned, and the door is opened and the reaction proceeds.
However, when an inhibitor which resembles the substrate is present, it will compete with
the substrate for the position in the enzyme lock. When the inhibitor wins, it gains the lock
position but is unable to open the lock. Hence, the observed reaction is slowed down
because some of the available enzyme sites are occupied by the inhibitor.
NON-COMPETITIVE INHIBITORS ARE CONSIDERED TO BE SUBSTANCES
WHICH WHEN ADDED TO THE ENZYME ALTER THE ENZYME IN A WAY
THAT IT CANNOT ACCEPT THE SUBSTRATE. FIGURE 10.
SUBSTRATE INHIBITION WILL SOMETIMES OCCUR WHEN EXCESSIVE
AMOUNTS OF SUBSTRATE ARE PRESENT. FIGURE 11 SHOWS THE
REACTION VELOCITY DECREASING AFTER THE MAXIMUM VELOCITY
HAS BEEN REACHED.
ADDITIONAL AMOUNTS OF SUBSTRATE ADDED TO THE REACTION
MIXTURE AFTER THIS POINT ACTUALLY DECREASE THE REACTION
RATE. THIS IS THOUGHT TO BE DUE TO THE FACT THAT THERE ARE SO
MANY SUBSTRATE MOLECULES COMPETING FOR THE ACTIVE SITES ON
THE ENZYME SURFACES THAT THEY BLOCK THE SITES (FIGURE 12) AND
PREVENT ANY OTHER SUBSTRATE MOLECULES FROM OCCUPYING
THEM.
ENZYMES OF CLINICAL SIGNIFICANCE
TABLE 13-2 LISTS THE COMMONLY ANALYZED ENZYMES,
INCLUDING
THEIR SYSTEMATIC NAMES AND CLINICAL SIGNIFICANCE.