Factors Affecting Enzyme Activity in Biochemistry
BIOCHEMISTY
BINITA RANI
ASSOCIATE PROFESSOR (DAIRY CHEMISTRY)
FACULTY OF DAIRY TECHNOLOGY
S.G.I.D.T., BVC CAMPUS,
P.O.- BVC, DIST.-PATNA-800014
Course No.-DTC-111, Credit Hours – 2 (1+1)
FACTORS AFFECTING
ENZYME ACTIVITY
An
enzyme assay
measures => conversion of
substrate => product
,
under conditions of
cofactors, pH and temperature
at which enzyme
is
optimally active
.
An enzyme is most
conveniently assayed
by:
measuring
rate of appearance of product
or
rate of disappearance of substrate
.
If substrate absorbs light at a specific wavelength => then changes in
concentration of these molecules can be measured by => following
change of absorbance
at this wavelength.
Typically this is carried out using
spectrophotometer
=>
since
absorbance is proportional to the rate of enzyme activity
in moles of
substrate used (or product formed) per unit time.
Enzyme Velocity
Rate of an enzyme catalyzed reaction
is => its velocity.
It is normally reported as
=> values at time zero
( V0 ; micromoles
/min) since the
rate is fastest =>
at the point where
no product is yet
present.
This is because =>
substrate concentration is greatest =>
before any
substrate has been transformed to product.
A typical plot of
product
formed against
time
for an enzyme
catalyzed reaction show =>
an initial period of rapid product
formation
which gives => the
linear portion
of the plot.
This is followed by a slowing down of the enzyme rate as
substrate is used up during the reaction
.
Rate of enzyme-catalyzed reaction with time
Enzyme Unit
Enzyme activity
may be expressed => as
μ mol of substrate
transformed per minute ( μ mol min-1).
Standard unit
of enzyme activity are =>
enzyme unit
and
katal
(kat).
Enzyme unit
is =>
amount of enzyme =>
which catalyze the
transformation of
1 μ mol
of substrate per minute
at 25oC under
optimal conditions for that enzyme.
Katal
is the
SI unit
of enzyme activity => defined as
=> that
catalytic activity => which will raise the rate of reaction by one
mole per second in a specified system.
1U = 16.67 nanokatal
.
The term
activity
refers to =>
total units of enzyme in the
sample, whereas
the specific activity
is =>
number of enzyme units per milligram
of protein (Units/mg).
Substrate Concentration
Normal pattern of dependence of
enzyme rate on => substrate
concentration
(S) is that,
at
low
substrate concentrations a doubling of (S) will lead to =>
doubling of initial velocity (V0).
However,
at
higher
substrate concentration
=>
enzyme becomes
saturated
, and
further increase in (S)
leads to =>
very small changes
in V0
called =>
Vmax
.
This occurs because => at
saturating substrate concentrations
effectively =>
all of the enzyme molecules have bound to substrate.
Overall rate
is now dependent on => rate at which
product can
dissociate from the enzyme
and
adding further substrate will not
affect this
.
Effect of substrate concentration on enzyme-catalyzed reaction
The shape of the resulting graph when V0 is plotted against (S) is
called
hyperbola curve
.
Effect of pH
Enzymes have an
optimum pH
(or pH range) at which => their activity
is maximal.
At
higher or lower pH
=>
activity decreases
.
Amino acid side chains => in the active site may act as
weak acids
and
bases
with critical functions
that depend on => their maintaining a
certain state of ionization
, and => elsewhere
in the protein ionized side
chains
=> may play an
essential role in the interactions
=> that
maintain protein structure.
Large deviations in pH
lead to => denaturation of enzyme protein
itself => due to interference with
many weak noncovalent bonds =>
maintaining the three - dimensional structure .
Effect of pH on enzyme-catalyzed reaction
A graph of V0 plotted against pH will usually give =>
a bell - shaped curve
.
Many enzyme have pH optimum of around
6.8
but => there is a
great diversity
in pH optima of enzyme due to =>
different environments
in which they are
adapted to work.
Enzyme Concentration
Where the
substrate concentration is saturating
(i.e all the
enzyme molecules are bound to substrate) =>
doubling the
enzyme concentration will lead to => doubling of V0
.
This gives =>
a straight-line graph
when V0 is plotted against
enzyme concentration.
Effect of Temperature
Raising
temperature =>
increases the rate
of
both uncatalyzed
and
enzyme-catalyzed reactions
by =>
increasing kinetic energy
and the
collision frequency
of =>
reacting molecules
.
However,
heat energy
can also increase =>
kinetic energy
of the
enzyme
to a point => that
exceeds the energy barrier
for =>
disrupting
noncovalent interactions
that maintain =>
enzyme’s three-
dimensional structure.
Polypeptide chain
then begins to =>
unfold, or denature
, with an
accompanying =>
rapid loss of catalytic activity
.
Temperature range
over which => an enzyme maintains =>
stable, catalytically competent conformation
depends upon—and
typically moderately exceeds—the
normal temperature
of the
cells => in which it resides.
Enzymes from humans
generally
exhibit stability
at =>
temperatures up to
45–55 °C
.
By contrast, enzymes from the
thermophilic microorganisms =>
reside in volcanic hot springs or undersea hydrothermal vents =>
may be stable up to or above
100 °C
.
Effect of Temperature on enzyme-catalyzed reaction
Isoenzymes
Isoenzymes
=>
different forms of an enzyme
which =>
catalyze
the
same reaction
=> but
exhibit different physical or kinetic
properties =>
such as
isoelectric point, pH optimum, substrate
affinity or effect of inhibitors.
Different
isoenzymes forms of a given enzyme are usually derived
from =>
different genes
and often occur in =>
different tissues of
body.
Functional
lactate dehydrogenase
are =>
homo or hetero
tetramers
composed of
M and H protein subunits =>
encoded
by the
LDHA
and
LDHB
genes,
respectively:
LDH-1 (4H) - in the
heart and RBCs
LDH-2 (3H1M) - in the
reticuloendothelial
system
LDH-3 (2H2M) - in the
lungs
LDH-4 (1H3M) - in the
kidneys, placenta and pancreas
LDH-5 (4M) - in the
liver and skeletal muscle
Five isoenzymes that are usually described in the literature =>
each contain =>
four subunits
.
The major isoenzymes of
skeletal muscle and liver
=>
M4
, has =>
four muscle (M) subunits
; while
H4 is the main isoenzymes for
heart muscle
in most species =>
containing
four heart (H) subunits
.
Other variants contain
=> both types of subunits
.
THANKS
Enzyme assays measure substrate conversion to product under varying conditions like cofactors, pH, and temperature. Enzyme velocity represents the rate of a catalyzed reaction, typically reported as V0. Enzyme activity is expressed as mol of substrate transformed per minute, with enzyme unit and katal being standard units. Katal is the SI unit of enzyme activity, defined as the catalytic activity increasing the reaction rate by one mole per second. Substrate concentration impacts enzyme rate, with low concentrations showing a nonlinear relationship with the enzyme rate.
Download Presentation
Please find below an Image/Link to download the presentation.
The content on the website is provided AS IS for your information and personal use only. It may not be sold, licensed, or shared on other websites without obtaining consent from the author. Download presentation by click this link. If you encounter any issues during the download, it is possible that the publisher has removed the file from their server.
E N D
Presentation Transcript
BIOCHEMISTY Course No.-DTC-111, Credit Hours 2 (1+1) FACTORS AFFECTING ENZYME ACTIVITY BINITA RANI ASSOCIATE PROFESSOR (DAIRY CHEMISTRY) FACULTY OF DAIRY TECHNOLOGY S.G.I.D.T., BVC CAMPUS, P.O.- BVC, DIST.-PATNA-800014
An enzyme assay measures => conversion of substrate => product, under conditions of cofactors, pH and temperature at which enzyme is optimally active. An enzyme is most conveniently assayed by: measuring rate of appearance of product or rate of disappearance of substrate. If substrate absorbs light at a specific wavelength => then changes in concentration of these molecules can be measured by => following change of absorbance at this wavelength. Typically this is carried out using spectrophotometer => since absorbance is proportional to the rate of enzyme activity in moles of substrate used (or product formed) per unit time.
Enzyme Velocity Rate of an enzyme catalyzed reaction is => its velocity. It is normally reported as => values at time zero ( V0 ; micromoles /min) since the rate is fastest => at the point where no product is yet present. This is because => substrate concentration is greatest => before any substrate has been transformed to product.
A typical plot of product formed against time for an enzyme catalyzed reaction show => an initial period of rapid product formation which gives => the linear portion of the plot. This is followed by a slowing down of the enzyme rate as substrate is used up during the reaction. Rate of enzyme-catalyzed reaction with time
Enzyme Unit Enzyme activity may be expressed => as mol of substrate transformed per minute ( mol min-1). Standard unit of enzyme activity are => enzyme unit and katal (kat). Enzyme unit is => amount of enzyme => which catalyze the transformation of 1 mol of substrate per minute at 25oC under optimal conditions for that enzyme.
Katal is the SI unit of enzyme activity => defined as => that catalytic activity => which will raise the rate of reaction by one mole per second in a specified system. 1U = 16.67 nanokatal. The term activity refers to => total units of enzyme in the sample, whereas the specific activity is => number of enzyme units per milligram of protein (Units/mg).
Substrate Concentration Normal pattern of dependence of enzyme rate on => substrate concentration (S) is that, at low substrate concentrations a doubling of (S) will lead to => doubling of initial velocity (V0). However, at higher substrate concentration => enzyme becomes saturated, and further increase in (S) leads to => very small changes in V0 called => Vmax. This occurs because => at saturating substrate concentrations effectively => all of the enzyme molecules have bound to substrate. Overall rate is now dependent on => rate at which product can dissociate from the enzyme and adding further substrate will not affect this.
The shape of the resulting graph when V0 is plotted against (S) is called hyperbola curve. Effect of substrate concentration on enzyme-catalyzed reaction
Effect of pH Enzymes have an optimum pH (or pH range) at which => their activity is maximal. At higher or lower pH => activity decreases. Amino acid side chains => in the active site may act as weak acids and bases with critical functions that depend on => their maintaining a certain state of ionization, and => elsewhere in the protein ionized side chains => may play an essential role in the interactions => that maintain protein structure. Large deviations in pH lead to => denaturation of enzyme protein itself => due to interference with many weak noncovalent bonds => maintaining the three - dimensional structure .
A graph of V0 plotted against pH will usually give => a bell - shaped curve. Many enzyme have pH optimum of around 6.8 but => there is a great diversity in pH optima of enzyme due to => different environments in which they are adapted to work. Effect of pH on enzyme-catalyzed reaction
Enzyme Concentration Where the substrate concentration is saturating (i.e all the enzyme molecules are bound to substrate) => doubling the enzyme concentration will lead to => doubling of V0. This gives => a straight-line graph when V0 is plotted against enzyme concentration.
Effect of Temperature Raising temperature => increases the rate of both uncatalyzed and enzyme-catalyzed reactions by => increasing kinetic energy and the collision frequency of => reacting molecules. However, heat energy can also increase => kinetic energy of the enzyme to a point => that exceeds the energy barrier for => disrupting noncovalent interactions that maintain => enzyme s three- dimensional structure. Polypeptide chain then begins to => unfold, or denature, with an accompanying => rapid loss of catalytic activity.
Temperature range over which => an enzyme maintains => stable, catalytically competent conformation depends upon and typically moderately exceeds the normal temperature of the cells => in which it resides. Enzymes from humans generally exhibit stability at => temperatures up to 45 55 C. By contrast, enzymes from the thermophilic microorganisms => reside in volcanic hot springs or undersea hydrothermal vents => may be stable up to or above 100 C.
Isoenzymes Isoenzymes => different forms of an enzyme which => catalyze the same reaction => but exhibit different physical or kinetic properties => such as isoelectric point, pH optimum, substrate affinity or effect of inhibitors. Different isoenzymes forms of a given enzyme are usually derived from => different genes and often occur in => different tissues of body.
Functional lactate dehydrogenase are => homo or hetero tetramers composed of M and H protein subunits => encoded by the LDHA and LDHB genes, respectively: LDH-1 (4H) - in the heart and RBCs LDH-2 (3H1M) - in the reticuloendothelial system LDH-3 (2H2M) - in the lungs LDH-4 (1H3M) - in the kidneys, placenta and pancreas LDH-5 (4M) - in the liver and skeletal muscle
Five isoenzymes that are usually described in the literature => each contain => four subunits. The major isoenzymes of skeletal muscle and liver => M4, has => four muscle (M) subunits; while H4 is the main isoenzymes for heart muscle in most species => containing four heart (H) subunits. Other variants contain => both types of subunits.
