Enzyme

Anita Kurniati
What are
enzymes?

Enzymes are
PROTEINS
(tertiary and
quaternary
structures).
• Enzymes are specific biologic proteins that catalyze biochemical
reactions without altering the equilibrium point of the reaction or
being consumed or changed in composition.
• The catalyzed reactions are frequently specific and essential to
physiologic functions, such as the hydration of carbon dioxide, nerve
conduction, muscle contraction, nutrient degradation, and energy
use.
• Found in all body tissue, enzymes frequently appear in the serum
following cellular injury or, sometimes, in smaller amounts, from
degraded cells.
• Certain enzymes, such as those that facilitate coagulation, are
specific to plasma and, therefore, are present in significant
concentrations in plasma.
• Plasma or serum enzyme levels are often useful in the diagnosis of
particular diseases or physiologic abnormalities.
Enzyme Classification
Factors That Influence Enzymatic Reactions
Substrate
• 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.
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.
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 on 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.
• In the laboratory, the pH for a reaction is carefully controlled at the
optimal pH by means of appropriate buffer solutions.
Temperature
• Increasing 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.
• Each enzyme functions optimally at a particular temperature, which
is influenced by other reaction variables, especially the total time for
the reaction.
• The optimal temperature is usually close to that of the physiologic
environment of the enzyme; however, some denaturation may occur
at the human physiologic temperature of 37°C.
• The rate of denaturation increases as the temperature increases and
is usually significant at 40° to 50°C.
• Because low temperatures render enzymes reversibly inactive, many
serum or plasma specimens for enzyme measurement are
refrigerated or frozen to prevent activity loss until analysis.
• Storage procedures may vary from enzyme to enzyme because of
individual stability characteristics.
• Repeated freezing and thawing, however, tends to denature protein
and should be avoided.
• Because of their temperature sensitivity, enzymes should be
analyzed under strictly controlled temperature conditions.
• Incubation temperatures should be accurate within 0.1°C.
• Laboratories usually attempt to establish an analysis temperature for
routine enzyme measurement of 25°, 30°, or 37°C.
• Attempts to establish a universal temperature for enzyme analysis
have been futile and, therefore, reference ranges for enzyme levels
may vary significantly among laboratories. In the United States,
however, 37°C is most commonly used.
Cofactors
• Cofactors are nonprotein entities that must bind to particular
enzymes before a reaction occurs.
• Common activators (inorganic cofactors) are metallic (Ca+2, Fe+2,
Mg+2, Mn+2, Zn+2, 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.
• Activators function by alternating the spatial configuration of the
enzyme for proper substrate binding, linking substrate to the
enzyme or coenzyme, or undergoing oxidation or reduction.
• 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.
• For example, NAD as a cofactor may be reduced to nicotinamide
adenine dinucleotide phosphate (NADP) in a reaction in which the
primary substrate is oxidized.
• Increasing coenzyme concentration will increase the velocity of an
enzymatic reaction in a manner synonymous with increasing
substrate concentration.
Inhibitors
• Enzymatic reactions may not progress normally if a particular
substance, an inhibitor, interferes with the reaction.
• Competitive inhibitors physically bind to the active site of an enzyme
and compete with the substrate for the active site.
• With a substrate concentration significantly higher than the
concentration of the inhibitor, the inhibition is reversible because
the substrate is more likely than the inhibitor to bind the active site
and the enzyme has not been destroyed.
 Two Types of Enzyme Inhibitors

1. COMPETITIVE
INHIBITOR
Chemicals that resemble
an enzyme’s normal
substrate and
compete with it for
the active site.

Reversible depending on
concentration of
inhibitor and
substrate.
EXAMPLE: The drug Antabuse is used to help alcoholics
quit drinking. Antabuse inhibits aldehyde oxidase, resulting
in the accumulation of acetaldehyde (say a-si-’tell-de-hide)
during the metabolism of alcohol. Elevated acetaldehyde
levels cause symptoms of nausea and vomiting.
• A noncompetitive inhibitor binds an enzyme at a place other than the
active site and may be reversible in the respect that some naturally
present metabolic substances combine reversibly with certain enzymes.
• Noncompetitive inhibition also may be irreversible if the inhibitor destroys
part of the enzyme involved in catalytic activity.
• Because the inhibitor binds the enzyme independently from the substrate,
increasing substrate concentration does not reverse the inhibition.
 Two Types of Enzyme Inhibitors

2. NON – COMPETITIVE
INHIBITOR

Do not enter active

site, but bind to
another part of the
enzyme, causing the
enzyme & active site to
change shape.

Usually reversible,
depending on
concentration of
inhibitor & substrate.

EXAMPLE: You may know that compounds containing

heavy metals such as lead, mercury, copper or silver
are poisonous. This is because ions of these metals
are non-competitive inhibitors for several enzymes.
• Uncompetitive inhibition is another kind of inhibition in which the
inhibitor binds to the ES complex—increasing substrate concentration
results in more ES complexes to which the inhibitor binds and, thereby,
increases the inhibition.
• The enzyme–substrate–inhibitor complex does not yield product.
• The substrate and inhibitor, commonly a metallic ion, may bind an
enzyme simultaneously in noncompetitive inhibition.
• The inhibitor may inactivate either an ES complex or just the enzyme by
causing structural changes in the enzyme.
• Even if the inhibitor binds reversibly and does not inactivate the enzyme,
the presence of the inhibitor when it is bound to the enzyme slows the
rate of the reaction.
• Thus, for noncompetitive inhibition, the maximum reaction velocity
cannot be achieved. Increasing substrate levels has no influence on the
binding of a noncompetitive inhibitor, so the Km is unchanged.
• Because uncompetitive inhibition requires the formation of an ES
complex, increasing substrate concentration increases inhibition.
Therefore, maximum velocity equal to that of an uninhibited reaction
cannot be achieved, and the Km appears to be decreased.
Measurement of Enzyme Activity
• Because enzymes are usually present in very small quantities in biologic
fluids and often difficult to isolate from similar compounds, a convenient
method of enzyme quantitation is measurement of catalytic activity.
• Activity is then related to concentration.
• Common methods might photometrically measure an increase in product
concentration, a decrease in substrate concentration, a decrease in
coenzyme concentration, or an increase in the concentration of an altered
coenzyme.
• NADH is a coenzyme frequently measured in the laboratory.
• NADH absorbs light at 340 nm, whereas NAD does not, and a change in
absorbance at 340 nm is easily measured.
• One of two general methods may be used to measure the extent of an
enzymatic reaction:
(1) fixed-time and
(2) continuous-monitoring or kinetic assay. In the fixedtime method, the
reactants are combined, the reaction proceeds for a designated time,
the reaction is stopped (usually by inactivating the enzyme with a weak
acid), and a measurement is made of the amount of reaction that has
occurred. The reaction is assumed to be linear over the reaction time;
the larger the reaction, the more enzyme is present.
 Calculation of Enzyme Activity
• When enzymes are quantitated relative to their activity rather than a direct
measurement of concentration, the units used to report enzyme levels are activity
units.
• The definition for the activity unit must consider variables that may alter results (e.g.,
pH, temperature, substrate).
• Historically, specific method developers frequently established their own units for
reporting results and often named the units after themselves (i.e., Bodansky and King
units).
• To standardize the system of reporting quantitative results, the EC defined the
international unit (IU) as the amount of enzyme that will catalyze the reaction of 1
mol of substrate per minute under specified conditions of temperature, pH,
substrates, and activators.
• Because the specified conditions may vary among laboratories, reference values are
still often laboratory specific. Enzyme concentration is usually expressed in units per
liter (IU/L).