Factors affecting EnzymeCatalyzed Reactions

Prof. Dr. Mohamed Fawzy Ramadan Hassanien

Zagazig University, Egypt

Factors affecting Enzyme-Catalyzed

Reactions
1- Effect of Substrate Concentration
(Single-Substrate Reactions)
2- Effect of Inhibitors
3- Effect of pH
4- Effect of Temperature
5- Effect of Pressure
6- Effect of Water

-Enzymes in food can be detected only indirectly by measuring

their catalytic activity and, in this way, differentiated from other
enzymes.
-This is the rationale for acquiring knowledge needed to analyze
the parameters which inuence or determine the rate of an
enzyme-catalyzed reaction.
-The reaction rate is dependent on the concentrations of the
components involved in the reaction.
-Here, it means primarily the substrate and the enzyme. Also, the
reaction can be inuenced by the presence of activators and
inhibitors.

Finally, the pH, the ionic strength of the reaction medium, the
dielectric constant of the solvent (usually water) and the
temperature exert an effect.

1 Effect of Substrate Concentration

Single-Substrate Reactions
-Let us consider a single-substrate reaction.
-Enzyme E reacts with substrate A to form an intermediary
enzyme-substrate complex, EA.
The complex then forms the product P and releases the free
enzyme:

-In order to determine the catalytic activity of the enzyme, the

decrease in substrate concentration or the increase in product
concentration as a function of time can be measured.

1 Effect of Substrate Concentration

Single-Substrate Reactions
The activity curve obtained has the
following regions:

a) The maximum activity which occurs

for a few msec until an equilibrium is
reached between the rate of enzymesubstrate formation and rate of
breakdown of this complex.
-Measurements in this pre-steady state
region which provide an insight into the
reaction steps and mechanism of catalysis
are difficult and time consuming.
-Hence, further analysis of the pre-steady
state should be ignored.

Progress of an
enzyme-catalyzed
reaction

b) The usual procedure is to measure the enzyme activity when a

steady state has been reached. In the steady state, the
intermediary complex concentration remains constant while the
concentration of the substrate and end product are changing.

C) The reaction rate decreases in this region in spite of an excess of

substrate.
The decrease in the reaction rate can be considered to be result of:

-Enzyme denaturation which can readily occur,

-Decreasing the enzyme concentration in the reaction system, or
the product formed increasingly inhibits enzyme activity or, after
the concentration of the product increases,
-The reverse reaction takes place, converting the product back into
the initial reactant.

2 Effect of Inhibitors
-The catalytic activity of enzymes, in addition to substrate
concentration, is affected by the type and concentration
of inhibitors, i.e. compounds which decrease the rate of
catalysis, and activators, which have the opposite effect.
-Activitors include
-metal ions and
-compounds which are active as prosthetic groups or
which provide stabilization of the enzymes conformation
or of the enzyme-substrate complex.
-Inhibitors are found among food constituents.

2 Effect of Inhibitors
-Based on kinetic considerations, inhibitors are divided
into two groups:
inhibitors bound irreversibly to enzyme and
inhibitors bound reversibly to enzyme .
-Proteins which specically inhibit the activity of certain
peptidases, amylases or -fructofuranosidase are
examples.
-Furthermore, food contains substances which nonselectively inhibit a wide spectrum of enzymes.
-Phenolic compounds of food and mustard oil belong to
this group.

2 Effect of Inhibitors
-Food might be contaminated with pesticides, heavy
metal ions and other chemicals from a polluted
environment which can become inhibitors under some
circumstances.
-These possibilities should be taken into account when
enzymatic food analysis is performed.

-Food is usually heat treated to suppress undesired

enzymatic reactions.
-As a rule, no inhibitors are used in food processing.
-An exception is the addition of, for example, SO2 to
inhibit the activity of phenolase.

2 Effect of Inhibitors
-Much data concerning the mechanism of action
of enzyme inhibitors have been published in
recent biochemical research.
These data cover
-the elucidation of the effect of inhibitors on
functional groups of an enzyme,
-the effect of inhibitors on the active site and
-the clarication of the general mechanism
involved in an enzyme catalyzed reaction.

3 Effect of pH on Enzyme Activity

-Each enzyme is active only in a narrow pH range and each has a
pH optimum which is often between pH 5.5 and 7.5 (Table).
-The optimum pH is affected by the type and ionic strength of the
buffer used in the assay.

pH Optima of various enzymes

3 Effect of pH on Enzyme Activity

The reasons for the sensitivity of the enzyme to changes in pH are:
a) Sensitivity is associated with a change in protein structure
leading to irreversible denaturation,
b) The catalytic activity depends on the quantity of electrostatic
charges on the enzymes active site generated by the prototropic
groups of the enzyme.
-In addition the ionization of dissociable substrates as affected by
pH can be of importance to the reaction rate.
-However, such effects should be determined separately.

4 Inuence of Temperature
-Thermal processes are important factors in the processing and
storage of food because they allow the control of chemical,
enzymatic and microbial changes.

-Undesired changes can be delayed or stopped by refrigerated

storage. Heat treatment may either accelerate desirable chemical or
enzymatic reactions or inhibit undesirable changes by inactivation
of enzymes or microorganisms.
-Temperature and time are two parameters responsible for the
effects of a thermal treatment.
-They should be selected carefully to make sure that all necessary
changes, e. g., killing of pathogens, are guaranteed, but still all
undesired changes such as degradation of vitamins are kept as low
as possible.
-The following table informs about quality deterioration caused by
enzymes which can be eliminated e. g., by thermal inactivation.

Thermal inactivation of enzymes to prevent

deterioration of food quality

4 Inuence of Temperature

Temperature Optimum
-Enzyme-catalyzed reactions and the growth of
microorganisms show a so-called temperature
optimum, which is a temperature-dependent
maximum resulting from the overlapping of two
counter effects with signicantly different
activation energies:

increase in reaction or growth rate

increase in inactivation or killing rate

4 Inuence of Temperature

Temperature Optimum
-For starch hydrolysis by microbial -amylase, the
activation energies were :

Ea (hydrolysis) = 20 kJ mol1
Ea (inactivation) = 295 kJ mol1
-As a consequence of the difference in activation
energies, the rate of enzyme inactivation is faster with
increasing temperature than the rate of enzyme catalysis.
-Based on activation energies for the above example, the
following relative rates are obtained (Table).
-Increasing temperature from 0 to 60 C increases the
hydrolysis rate by a factor of 5, while rate of inactivation
is accelerated by more than 10 powers of ten.

Temperature Optimum
-Amylase activity as affected by temperature: relative
rates of hydrolysis and enzyme inactivation

Temperature Optimum
-The growth of microorganisms
follows a similar temperature
dependence and can also be
characterized by replacing the
value k by the growth rate and
assuming Ea is the reference
value of the temperature for
growth (Figure).
-For maintaining food quality,
detailed knowledge of the
relationship between microbial
growth rate and temperature is
important
for
optimum
production processes (heating,
cooling, freezing).

Growth rate and temperature for

1) psychrophilic (Vibrio AF-1),
2) mesophilic (E. coli) and
3) thermophilic (Bacillus cereus)
microorganisms

Temperature Optimum
-The highly differing activation energies for
killing microorganisms and for normal chemical
reactions have triggered a trend in food
technology towards the use of high-temperature
short-time (HTST) processes in production.
-These are based on the ndings that at higher
temperatures the desired killing rate of
microorganisms is higher than the occurrence of
undesired chemical reactions.

Thermal Stability
-The thermal stability of enzymes is quite
variable.
-Some enzymes lose their catalytic activity
at lower temperatures, while others are
capable of withstanding (at least for a short
period of time) a stronger thermal
treatment.
-In a few cases enzyme stability is lower at
low temperatures than in the medium
temperature range.

Thermal Stability
-Lipase and alkaline
phosphatase in milk are
thermolabile, whereas
acid phosphatase is
relatively stable (Figure).

-Therefore,
alkaline
phosphatase is used to
distinguish raw from Thermal inactivation of enzymes of milk.
1 Lipase (inactivation extent, 90%),
pasteurized
milk
2 alkaline phosphatase (90%),
because its activity is
3 catalase (80%),
4 xanthine oxidase (90%),
easier to determine than
5 peroxidase (90%), and
that of lipase.
6 acid phosphatase (99%)

Thermal Stability
-Of all the enzymes in the potato tuber (Figure),
peroxidase is the last one to be thermally inactivated.

Thermal inactivation (90%) of enzymes

present in potato tuber

Thermal Stability
-Such inactivation patterns are often found among
enzymes in vegetables. In such cases, peroxidase is a
suitable indicator for controlling the total inactivation of
all the enzymes e.g., in assessing the adequacy of a
blanching process.
-However, newer developments aim to limit the enzyme
inactivation to such enzymes responsible for quality
deterioration during storage.
-For example pea seeds in which lipoxygenase is
responsible for spoilage. However, lipoxygenase is more
sensitive than peroxidase, thus a sufcient but gentle
blanching requires the inactivation of lipoxygenase only.
Inactivation of peroxidase is not necessary.

5 Inuence of Pressure
-The application of high pressures can inhibit the growth of
microorganisms and the activity of enzymes. This allows the
protection of sensitive nutrients and aroma substances in foods.

-Some products preserved in this gentle way are now in the

market.
-Microorganisms are relatively sensitive to high pressure because
their growth is inhibited at pressures of 300600 MPa and lower
pH values increase this effect. However, bacterial spores withstand
pressures of >1200 MPa.
-In contrast to thermal treatment, high pressure does not attack
the primary structure of proteins at room temperature. Only Hbridges, ionic bonds and hydrophobic interactions are disrupted.
Quaternary structures are dissociated into subunits by
comparatively low pressures (<150 MPa).

5 Inuence of Pressure
-Higher pressures (>1200 MPa) change the tertiary structure and
very high pressures disrupt the H-bridges which stabilize the
secondary structure.
-The hydration of proteins is also changed by high pressure
because water molecules are pressed into cavities which can exist
in the hydrophobic interior of proteins.
-In general, proteins are irreversibly denatured at room
temperature by the application of pressures above 300 MPa while
lower pressures cause only reversible changes in the protein
structure.

5 Inuence of
Pressure
-In the case of enzymes, even
slight changes in the steric
arrangement and mobility of the
amino acid residues which
participate in catalysis can lead to
loss of activity.
-Nevertheless, a relatively high
pressure is often required to
inhibit enzymes. But the pressure
required can be reduced by Pressure-temperature diagram for
the inactivation kinetics of increasing the temperature, as
shown in the Figure for - amylase from Bacillus subtilis at
amylase.
pH 8.6.

5 Inuence of Pressure
-While a pressure of 550 MPa is required at 25 C to inactivate
the enzyme, a pressure of only 340 MPa is required at 50 C.
-It is remarkable that enzymes can also be activated by changes in
the conformation of the polypeptide chain, which are initiated
especially by low pressures around 100 MPa.
-In the application of the pressure technique for the production of
stable food, intact tissue, and not isolated enzymes, is exposed to
high pressures.

-Thus, the enzyme activity can increase instead of decreasing when

cells or membranes are disintegrated with the release of enzyme
and/or substrate.

5 Inuence of Pressure
-Some examples are presented here to show the pressures
required to inhibit the enzyme activity which can negatively effect
the quality of foods.

Pectin methylesterase: causes the occulation of pectic acid in

orange juices and reduces the consistency of tomato products. In
orange juice, irreversible enzyme inactivation reaches 90% at a
pressure of 600 MPa. Even though the enzyme in tomatoes is more
stable, increasing the temperature to 5960 C causes inactivation
at 400 Mpa and at 100 MPa after the removal of Ca2+ ions.
Peroxidases: induce undesirable aroma changes in plant foods. In
green beans, enzyme inactivation reached 88% in 10 min after
pressure treatment at 900 MPa. At pressures above 400 MPa (32
C), the activity of this enzyme in oranges fell continuously to 50%.
However, very high pressures increased the activity at 3260 C.

5 Inuence of Pressure
-It is possible that high pressure denatures peroxidase to a
heme(in) catalyst to 50%. However, very high pressures increased
the activity at 3260 C. It is possible that high pressure
denatures peroxidase to a heme(in) catalyst.
Lipoxygenase from soybeans.
This enzyme was inactivated in 5 min at pH 8.3 by pressures up
to 750 MPa and temperatures in the range 075 C. The pressure
stability was reduced by gassing with CO2 and reducing pH to 5.4.
Polyphenol oxidases in mushrooms and potatoes require
pressures of 800900 Mpa for inactivation. The addition of
glutathione (5 mmol/l) increases the pressure sensitivity of the
mushroom enzyme. In this case, the inactivation is obviously
supported by the reduction of disulde bonds.

6 Inuence of
Water
-Up to certain extent,
enzymes need to be hydrated
in order to develop activity.
-Hydration of e.g. lysozyme
was determined by IR and
NMR spectroscopy.
-As can be seen in Table, rst
the charged polar groups of
the side chains hydrate,
followed by the uncharged
ones.

Hydration of Lysozyme

6 Inuence of Water
-Enzymatic activity starts at a water content of 0.2 g/g
protein, which means even before a monomolecular layer
of the polar groups with water has taken place.
-Increase in hydration resulting in a monomolecular layer of
the whole available enzyme surface at 0.4 g/g protein raises
the activity to a limiting value reached at a water content of
0.9 g/g protein. Here the diffusion of the substrate to the
enzymes active site seems to be completely guaranteed.

-For preservation of food it is mandatory to inhibit

enzymatic activity completely if the storage temperature is
below the phase transition temperature.