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Enzyme catalysis and inhibition
Enzyme catalysis: Free energy is released by the formation of a large number of weak
interactions between a complementary enzyme and its substrate. The free energy released
on binding is called the binding energy. Only the correct substrate can participate in most or
all of the interactions with the enzyme and thus maximize binding energy, accounting for the
exquisite substrate specificity exhibited by many enzymes. Furthermore, the full complement
of such interactions is formed only when the substrate is converted into the transition state.
Thus, the maximal binding energy is released when the enzyme facilitates the formation
of the transition state. The energy released by the interaction between the enzyme and the
substrate can be thought of as lowering the activation energy.
The lineweaver-burk transformation
The Km and Vmax for an enzyme can be visually determined from a plot of 1/V versus 1/[S],
called a lineweaver-burk or a double reciprocal plot.
The reciprocal of both sides of the Michaelis-Menten equation generates an equation that has
the form of a straight line, y = mx+ c. Km and Vmax are equal to the reciprocals of the intercepts
on the x-axis and y-axis, respectively. The purpose of this transformation make extrapolation
easier so one can obtain accurate assessments of the Km and Vmax.
Turnover number: The maximal velocity Vmax reveals the turnover number of an enzyme,
which is the number of substrate molecules that an enzyme can convert into a product per unit
time when the enzyme is fully saturated with substrate. The turnover number is equal to the
rate constant k2, which is also called kcat. If the total concentration of active sites, [E]T, is
known, then V max = k2 [E]T and thus k2 = Vmax / [E]T. Turnover numbers for most enzymes
are between 1 and 104 per second.
The rate constant kcat /KM called the specificity constant, is a measure of catalytic efficiency
because it takes into account both the rate of catalysis with a particular substrate (kcat) and the
nature of the enzyme-substrate interaction (KM). The upper limit on kcat /KM is between 108
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and 109 s −1 M −1
. Enzymes that have kcat /KM ratios at the upper limits have attained kinetic
perfection.
Enzyme specific activity refers to the activity of an enzyme per milligram of total protein.
It is a measure of the purity of an enzyme preparation. Specific activity is often used to assess
the degree to which an enzyme has been purified from a crude extract. The specific activity of
an enzyme is calculated using the following formula: Specific activity = Enzyme activity
(units) /Total protein (mg)
Enzyme activity (units): This is the measure of the enzymatic activity, usually expressed in
units. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the
conversion of 1 micromole of substrate per minute under standard assay conditions.
nkat (nanokatal): A "nkat" (nanokatal) is a metric unit of enzyme activity in the International
System of Units (SI). One nanokatal is equal to 1 × 10-9 katal. A katal is defined as the amount
of enzyme that can convert 1 mole of substrate per second.
Enzyme inhibition
Enzyme inhibition can be either reversible or irreversible. We begin the investigation of
enzyme inhibition by first examining reversible inhibition. In contrast with irreversible
inhibition, reversible inhibition is characterized by rapid dissociation of the enzyme-inhibitor
complex. There are three common types of reversible inhibition: competitive inhibition,
uncompetitive inhibition, and non-competitive inhibition. These three types of inhibition differ
in the nature of the interaction between the enzyme and the inhibitor and in the inhibitor’s effect
on enzyme kinetics. Double-reciprocal plots are especially useful for distinguishing
competitive, uncompetitive, and noncompetitive inhibitors.
Whereas a reversible inhibitor will both bind to an enzyme and dissociate from it rapidly, an
irreversible inhibitor dissociates very slowly from its target enzyme because it has become
tightly bound to the enzyme, either covalently or noncovalently.
Irrevervisble inhibition
Competitive inhibition: A substance that competes directly with a normal substrate for an
enzymatic binding site is known as a competitive inhibitor. Such an inhibitor usually resembles
the substrate to the extent that it specifically binds to the active site but differs from it to be
unreactive. Competitive inhibitors increase the apparent Km of the enzyme for the substrate.
This means that a higher substrate concentration is required to achieve half of Vmax. The
competitive inhibitor competes with the substrate for binding to the active site of the enzyme.
As a result, a higher substrate concentration is needed to outcompete the inhibitor and
achieve the same rate of reaction as in the absence of the inhibitor. This is reflected by an
Enzyme catalysis and inhibition
Enzyme catalysis: Free energy is released by the formation of a large number of weak
interactions between a complementary enzyme and its substrate. The free energy released
on binding is called the binding energy. Only the correct substrate can participate in most or
all of the interactions with the enzyme and thus maximize binding energy, accounting for the
exquisite substrate specificity exhibited by many enzymes. Furthermore, the full complement
of such interactions is formed only when the substrate is converted into the transition state.
Thus, the maximal binding energy is released when the enzyme facilitates the formation
of the transition state. The energy released by the interaction between the enzyme and the
substrate can be thought of as lowering the activation energy.
The lineweaver-burk transformation
The Km and Vmax for an enzyme can be visually determined from a plot of 1/V versus 1/[S],
called a lineweaver-burk or a double reciprocal plot.
The reciprocal of both sides of the Michaelis-Menten equation generates an equation that has
the form of a straight line, y = mx+ c. Km and Vmax are equal to the reciprocals of the intercepts
on the x-axis and y-axis, respectively. The purpose of this transformation make extrapolation
easier so one can obtain accurate assessments of the Km and Vmax.
Turnover number: The maximal velocity Vmax reveals the turnover number of an enzyme,
which is the number of substrate molecules that an enzyme can convert into a product per unit
time when the enzyme is fully saturated with substrate. The turnover number is equal to the
rate constant k2, which is also called kcat. If the total concentration of active sites, [E]T, is
known, then V max = k2 [E]T and thus k2 = Vmax / [E]T. Turnover numbers for most enzymes
are between 1 and 104 per second.
The rate constant kcat /KM called the specificity constant, is a measure of catalytic efficiency
because it takes into account both the rate of catalysis with a particular substrate (kcat) and the
nature of the enzyme-substrate interaction (KM). The upper limit on kcat /KM is between 108
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and 109 s −1 M −1
. Enzymes that have kcat /KM ratios at the upper limits have attained kinetic
perfection.
Enzyme specific activity refers to the activity of an enzyme per milligram of total protein.
It is a measure of the purity of an enzyme preparation. Specific activity is often used to assess
the degree to which an enzyme has been purified from a crude extract. The specific activity of
an enzyme is calculated using the following formula: Specific activity = Enzyme activity
(units) /Total protein (mg)
Enzyme activity (units): This is the measure of the enzymatic activity, usually expressed in
units. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the
conversion of 1 micromole of substrate per minute under standard assay conditions.
nkat (nanokatal): A "nkat" (nanokatal) is a metric unit of enzyme activity in the International
System of Units (SI). One nanokatal is equal to 1 × 10-9 katal. A katal is defined as the amount
of enzyme that can convert 1 mole of substrate per second.
Enzyme inhibition
Enzyme inhibition can be either reversible or irreversible. We begin the investigation of
enzyme inhibition by first examining reversible inhibition. In contrast with irreversible
inhibition, reversible inhibition is characterized by rapid dissociation of the enzyme-inhibitor
complex. There are three common types of reversible inhibition: competitive inhibition,
uncompetitive inhibition, and non-competitive inhibition. These three types of inhibition differ
in the nature of the interaction between the enzyme and the inhibitor and in the inhibitor’s effect
on enzyme kinetics. Double-reciprocal plots are especially useful for distinguishing
competitive, uncompetitive, and noncompetitive inhibitors.
Whereas a reversible inhibitor will both bind to an enzyme and dissociate from it rapidly, an
irreversible inhibitor dissociates very slowly from its target enzyme because it has become
tightly bound to the enzyme, either covalently or noncovalently.
Irrevervisble inhibition
Competitive inhibition: A substance that competes directly with a normal substrate for an
enzymatic binding site is known as a competitive inhibitor. Such an inhibitor usually resembles
the substrate to the extent that it specifically binds to the active site but differs from it to be
unreactive. Competitive inhibitors increase the apparent Km of the enzyme for the substrate.
This means that a higher substrate concentration is required to achieve half of Vmax. The
competitive inhibitor competes with the substrate for binding to the active site of the enzyme.
As a result, a higher substrate concentration is needed to outcompete the inhibitor and
achieve the same rate of reaction as in the absence of the inhibitor. This is reflected by an
