Lecture 7.1-Enzymes

23
Enzymes
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Lecture 8
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23-1
23 Enzyme Catalysis
• Enzyme: a biological catalyst.
• With the exception of some RNAs (Ribozymes) that
catalyze their own self-cleavage, all enzymes are
proteins (majority are globular).
1) Enzymes can increase the rate of a reaction by a
factor of 109 to 1020 over an uncatalyzed reaction.
• Like all catalysts, enzymes do not change the position of
equilibrium.
• They cause reactions to take place faster by lowering the activation
energy.
2) Most of them are extremely specific.

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23-2
23 Enzyme Catalysis
• Enzyme: a biological catalyst.
• Some catalyze the reaction of only one compound.
( NH2 )C=O + H2 O ureas e 2 NH3 + CO2
Urea
• Others are stereospecific; for example, enzymes that
catalyze the reactions of only L-amino acids.
• Others catalyze reactions of specific types of
compounds or bonds; for example, trypsin that
catalyzes hydrolysis of peptide bonds formed by the
carboxyl groups of Lys and Arg.

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23-3
23 Classification of Enzymes
• Enzymes are commonly named after the reaction
or reactions they catalyze and/or the compound
or type of compound on which they act.
• example: lactate dehydrogenase, acid phosphatase.
• Enzymes are classified into six major groups.
• Oxidoreductases: catalyze oxidation-reduction reactions.
• Transferases: catalyze group transfer reactions.
• Hydrolases: catalyze hydrolysis reactions.
• Lyases: catalyze the addition of groups to a double bond,
or removal of groups to create a double bond.
• Isomerases: catalyze isomerization reactions.
• Ligases (or synthetases): catalyze the joining of two
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molecules. 23-4
1. Oxidoreductase:
2. Transferase:
COO- COO- COO- COO-
Aspartate amino
CH2 C= O CH2 C-N H3 +
transferase
CH- NH3 + + CH2 or aspartate
C= O + CH2
COO- CH2 transaminase COO- CH2
COO- COO-
Aspartate -Ketoglutarate Oxalosuccinate Glutamate
3. Hydrolase:
O O
CH3 -C- OCH 2 CH 2 N( CH3 ) 2 + H2 O Acetylcholinesterease CH3 -C- O- HOCH2 CH2 N( CH3 ) 2
Acetylcholine Acetate Choline
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23-5
4. Lyase: COO- COO-
CH2 CH2
C-COO - + H O Aconitase C-COO -
2
CH HO C-H
COO- COO-
cis- Aconitate Isocitrate
5. Isomerase: CH2 OPO 3 2 - CH2 OPO 3 2 -

O Phosphohexose O CH2 OH
isomerase H HO
OH
HO OH H OH
OH HO H
 - D- Glucose-6-phosphate  - D-Fructose-6-phosphate
6. Ligase: Tyrosine-tRNA
synthetase L-tyrosyl-tDNA + AMP + PP
ATP + L-tyrosine + t-RNA i

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23-6
23 Terms in Enzyme Chem
• Apoenzyme: the protein part of an enzyme.
• Cofactor: a nonprotein portion of an enzyme that is
necessary for catalytic function; examples are metallic
ions such as Zn2+ and Mg2+.
• Coenzyme: a nonprotein organic molecule, frequently a
B vitamin, that acts as a cofactor.
o Another important coenzyme is heme, which is part of several
oxidoreductases as well as part of hemoglobin.
Apoenzyme + Coenzyme (co-factor) = Holoenzyme
• Substrate: the compound or compounds whose
reaction an enzyme catalyzes.
• Active site: the specific portion of the enzyme to which
a substrate binds during reaction.
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23-7
23 Schematic of an Active Site
• Figure 23.2 Schematic diagram of the active site of an
enzyme and the participating components.
In any case, an apoenzyme

cannot catalyze a reaction
If the enzyme has without its cofactor, nor can
coenzymes, they are the cofactor function without
located at the active site. the apoenzyme. When a
Therefore, the substrate metal ion is a cofactor, it can
is simultaneously
bind directly to the protein or
surrounded by parts of
the apoenzyme, to the coenzyme, if the
coenzyme, and metal ion enzyme contains one.
cofactor (if any).

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23-8
23 Terms in Enzyme Chem
• Activation: any process that initiates or increases the
activity of an enzyme.
o addition of a cofactor to an apoenzyme or the cleavage of a
polypeptide chain of a proenzyme (inactive enzyme)
• Inhibition: any process that makes an active enzyme less
active or inactive.
• Competitive inhibitor: any substance that binds to the

active site of an enzyme thereby preventing binding of
substrate.
• Noncompetitive inhibitor: any substance that binds to a
portion of the enzyme other than the active site and thereby
inhibits the activity of the enzyme.
Both competitive and noncompetitive inhibition are reversible, but some compounds
alter the structure of the enzyme permanently and thus make it irreversibly inactive.
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23-9
23 Enzyme Activity
• Enzyme activity: a measure of how much a
reaction rate is increased.
• We examine how the rate of an enzyme-catalyzed reaction
is affected by:
1. enzyme concentration
2. substrate concentration
3. Temperature
4. pH

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23-10
23 Enzyme Activity
• Figure 23.3 The effect of enzyme concentration on the rate
of an enzyme-catalyzed reaction. Substrate concentration,
temperature, and pH are constant.
If we keep the concentration of
substrate constant and increase the
concentration of enzyme, the rate
increases linearly (Fig 23.3). That is,
if the enzyme concentration doubles,
the rate doubles as well; if the
enzyme concentration triples, the
rate also triples. This is the case in
practically all enzyme reactions,
because the molar concentration of
enzyme is almost always much
lower than that of substrate (that is,
many more molecules of substrate
are typically present than molecules
of enzyme).
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23-11
23 Enzyme Activity
• Figure 23.4 The effect of substrate concentration on the
rate of an enzyme-catalyzed reaction. Enzyme
concentration, temperature, and pH are constant.
Conversely, if we keep the
concentration of enzyme constant
and increase the concentration of
substrate, we get a different type of
curve called a saturation curve
(Fig 23.4). In this case, the rate
does not increase continuously.
Instead, a point is reached after
which the rate stays the same even
if we increase the substrate
concentration further. This happens
because at the saturation point,
substrate molecules are bound to all
available active sites of the
enzymes.
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23-12
23 Enzyme Activity
• Figure 23.5 The effect of temperature on the rate of an
enzyme-catalyzed reaction. Substrate and enzyme
concentrations and pH are constant.
Temperature affects enzyme activity
because it changes the conformation of
the enzyme. In uncatalyzed reactions,
the rate usually increases as the
temperature increases. Changing the
temperature has a different effect on
enzyme-catalyzed reactions. When we
start at a low temperature (Fig 23.5), an
increase in temperature first causes an
increase in rate. However, protein
conformations are very sensitive to
temperature changes. Once the optimal
temperature is reached, any further
increase in temperature alters the
enzyme conformation. The substrate
may then not fit properly onto the
© 2006 Thomson Learning, Inc. changed enzyme surface, so the rate of
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reaction actually decreases. 23-13
23 Enzyme Activity
Figure 23.5 The effect of temperature
on the rate of an enzyme-catalyzed
reaction. Substrate and enzyme After a small temperature increase above the optimum, the
concentrations and pH are constant. decreased rate could be increased again by lowering the
temperature because over a narrow temperature range,
changes in conformation are reversible. However, at some
higher temperature above the optimum, we reach a point where
the protein denatures; the three-dimensional conformation
is then altered irreversibly, and the polypeptide chain
cannot refold to its native conformation. At this point, the
enzyme is completely inactivated. The inactivation of
enzymes at low temperatures is used in the preservation of
food by refrigeration.
Most enzymes from bacteria and higher organisms have an optimal temperature around
37°C. However, the enzymes of organisms that live at the ocean floor at 2°C have an optimal
temperature in that range. Other organisms live in ocean vents under extreme conditions,
and their enzymes have optimal conditions at ranges from 90 to 105°C. The enzymes of
these hyperthermophile organisms also have other extreme requirements, such as
pressures up to 100 atm, and some of them have an optimal pH in the range of 1 to 4.
Enzymes from these hyperthermophiles, especially polymerases that catalyze the
polymerization of DNA, have gained commercial importance.
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23-14
23 Enzyme Activity
• Figure 23.6 The effect of pH on the rate of an enzyme-
catalyzed reaction. Substrate and enzyme concentrations
and temperature are constant.
As the pH of its environment
changes the conformation of a
protein, we would expect pH-
related effects to resemble those
observed when the temperature
changes. Each enzyme operates
best at a certain pH (Fig 23.6).
Once again, within a narrow pH
range, changes in enzyme activity
are reversible. However, at
extreme pH values (either acidic or
basic), enzymes are denatured
irreversibly and enzyme activity
cannot be restored by changing
back to the optimal pH.
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23-15
23 Mechanism of Action
Arrhenius suggested that catalysts speed up reactions by combining
with the substrate to form some kind of intermediate compound. In an
enzyme-catalyzed reaction, this intermediate is called the enzyme–
substrate complex.
A. Lock-and-key model
• first model to explain the action of enzymes
• The enzyme is a rigid three-dimensional body.
• The enzyme surface contains the active site.
An enzyme molecule is very large (typically consisting of 100 to 200 amino acid
residues), but the active site is usually composed of only two or a few amino acid
residues, which may well be located at different places in the chain. This
arrangement emphasizes that the shape and the functional groups on the
surface
All of the active site are of importance in recognizing a substrate.
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23-16
B. Induced-fit model
• Introduced by American biochemist,
Daniel Koshland
• The active site becomes modified to
accommodate the substrate.

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23-17
• Figure 23.9 The mechanism of competitive inhibition.
Both the lock-and-key and induced-

fit models explain the phenomenon
of competitive inhibition. The
inhibitor molecule fits into the
active site cavity in the same
way the substrate does (Fig
23.9), thereby preventing the
substrate from entering. The
result: Whatever reaction is
supposed to take place on the
substrate does not occur.

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23-18
• Figure 23.10 Mechanism of noncompetitive inhibition.
Many cases of noncompetitive inhibition

can be explained by the induced-fit
model. The inhibitor does not bind
to the active site, but rather, binds
to a different part of the enzyme.
Nevertheless, the binding causes
a change in the three-dimensional
shape of the enzyme molecule,
which so alters the shape of the
active site that once the substrate
is bound, there can be no
catalysis (Fig 23.10).

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23-19
• Figure 23.11 Enzyme kinetics in the presence and absence
of inhibitors.
If we compare enzyme activity

in the presence and the
absence of an inhibitor, we can
tell whether competitive or
noncompetitive inhibition is
taking place (Fig 23.11).

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23-20
Figure 23.11 Enzyme kinetics in the presence and absence of inhibitors.
The maximum reaction rate is the same
without an inhibitor and in the presence of a
competitive inhibitor. The only difference is that
this maximum rate is achieved at a low substrate
concentration with no inhibitor but at a high
substrate concentration when an inhibitor is
present. This is the true sign of competitive
inhibition, because the substrate and the inhibitor
are competing for the same active site. If the
substrate concentration is sufficiently increased,
the inhibitor will be displaced from the active site
by Le Chatelier’s principle, thus allowing for
increased binding of the substrate and an
increased rate of reaction.
If the inhibitor is noncompetitive, it cannot be displaced by addition of excess substrate

because it is bound to a different site that is unaffected by the addition of excess substrate. In
this case, the enzyme cannot be restored to its maximum activity and the maximum rate of the
reaction is lower than it would be in the absence of the inhibitor.
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23-21
• Both the lock-and-key model and the induced-fit model
emphasize the shape of the active site.
• However, the chemistry of the active site is the most
important.
• Just five amino acids participate in the active sites in
more than 65% of the enzymes studies to date.
• These five are His > Cys > Asp > Arg > Glu.
• Four of these amino acids have either acidic or basic
side chains; the fifth has a sulfhydryl group (-SH).

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23-22
23 Catalytic Power
• Enzymes cannot change the thermodynamic
relationships between the substrates and the
products of a reaction; rather, they speed up the
reaction. But how do they really accomplish this
feat?

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23-23
23 Catalytic Power
• Enzymes provide an alternative pathway for reaction, one with a
significantly lower activation energy and, therefore, a faster rate.
Figure 23.12 (a) The activation energy profile for a typical reaction. (b) A comparison of
the activation energy profiles for catalyzed and uncatalyzed reactions.
The thermodynamic relationship is
described by the height difference
between the reactants and products,
as shown in Fig 23.12(a). In any
reaction:
A+B⟶C+D
before A and B can become C and D,
they must pass through a transition
state where they are something in
between. This situation is often
thought of as being an “energy hill”
that must be scaled. The energy
required to climb this hill is the
activation energy. Enzymes are
powerful catalysts because they
lower the energy hill, as shown in Fig
23.12(b). They reduce the activation
energy.
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23-24
23 Enzyme Regulation
• Enzymes are often regulated by environmental
conditions.
A. Feedback control: an enzyme-regulation process
where the product of a series of enzyme-catalyzed
reactions inhibits an earlier reaction in the sequence.
feedback inhibition
E1 E2 E3
A B C D
• The inhibition may be competitive or noncompetitive.

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23-25
Feedback control
The final product in the chain may inhibit the activity of

the first enzyme (by competitive, noncompetitive, or
some other type of inhibition). When the concentration
of the final product is low, all of the reactions proceed
rapidly. As the concentration increases, however, the
action of enzyme 1 becomes inhibited and eventually
stops. In this manner, the accumulation of the final
product serves as a message that tells enzyme 1 to
shut down because the cell has enough final product
for its present needs. Shutting down enzyme 1 stops
the entire process.
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23-26
B. Proenzyme (zymogen): an inactive form of an
enzyme that must have part of its polypeptide chain
hydrolyzed and removed before it becomes active.
• An example is trypsin, a digestive enzyme.

• It is synthesized and stored as trypsinogen (a zymogen), which
has no enzyme activity.
• It becomes active only after a six-amino acid fragment is hydrolyzed and
removed from the N-terminal end of its chain.
• Removal of this small fragment changes in not only the primary structure
but also the tertiary structure, allowing the molecule to achieve its active
form.
• The pancreas makes trypsin in an inactive form; only after trypsin enters
the digestive
© 2006 Thomson Learning, Inc. tract does it become active.
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23-27
C. Allosterism: enzyme regulation based on an
event occurring at a place other than the active site
but that creates a change in the active site.
• An enzyme regulated by this mechanism is called an
allosteric enzyme. If a substance binds noncovalently and
reversibly to a site other than the active site, it may affect the
enzyme in either of two ways:
• Negative modulation: inhibition of an allosteric enzyme.
• Positive modulation: stimulation of an allosteric enzyme.
• Regulator: a substance that binds to an allosteric
enzyme, and the site to which it attaches is called a regulatory
site. In most cases, allosteric enzymes contain more than one
polypeptide chain (subunits); the regulatory site is on one polypeptide
© 2006 Thomson chain,
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Learning,and
Inc. the active site is on another.
23-28
23 The Allosteric Effect
Figure 23.14 The allosteric effect. Binding of the regulator to
a site other than the active site changes the shape of the
active site.
In this case, the enzyme has only
one polypeptide chain, so it
carries both the active site and
the regulatory site at different
points in this chain. The regulator
binds reversibly to the regulatory
site. As long as the regulator
remains bound to the regulatory
site, the total enzyme–regulator
complex will be inactive. When
the regulator is removed from the
regulatory site, the enzyme
becomes active. In this way, the
regulator controls the
allosteric enzyme action.
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23-29
• Allosteric enzymes have two kinetic states, with a
corresponding conformational change in each of these states
that results in two different forms. One form is more likely to
bind the substrate and produce the product than the other
form. This more active form is referred to as the R form, where
“R” stands for relaxed. The less active form is referred to as
the T form, where “T” stands for taut (Fig 23.15). There is an
equilibrium between the T form and the R form. When the
enzyme is in the R form, it will bind substrate well and catalyze
the reaction. Allosteric regulators are seen to function by
binding to the enzyme and favoring one form versus the other.

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23-30
Figure 23.15 Effects of binding activators and inhibitors to
allosteric enzymes. The enzyme has an equilibrium between the T form
and the R form. An activator is anything that binds to the regulatory site
and favors the R form. An inhibitor binds to the regulatory site and favors
the T form.

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23-31
D. Protein modification: the process of affecting
enzyme activity by covalently modifying it.
• The modification is usually a change in the primary
structure, typically by addition of a functional group
covalently bound to the apoenzyme.
• The best known examples of protein modification
involve phosphorylation/dephosphorylation.
• Example: A phosphate group is often bonded to a serine or
tyrosine residue. In some enzymes, such as glycogen
phosphorylase, the phosphorylated form is the active form of
the enzyme. Without it, the enzyme is less active or inactive.

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23-32
D. Protein modification: the process of affecting
enzyme activity by covalently modifying it.
• The opposite example is the enzyme pyruvate kinase (PK).
Pyruvate kinase from the liver is inactive when it is phosphorylated.
Enzymes that catalyze such phosphorylation go by the common name of
kinases. When the activity of PK is not needed, it is phosphorylated (to
PKP) by a protein kinase using ATP as a substrate as well as a source of
energy. When the system wants to turn on PK activity, the phosphate
group, Pi, is removed by another enzyme, phosphatase, which renders
PK active.
ATP AD P
active inactive
kinase
PK PKP
phosphatase
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E. Isoenzyme: an enzyme that occurs in multiple forms
in different tissues; each catalyzes the same reaction.
• Example: lactate dehydrogenase (LDH) catalyzes the

oxidation of lactate to pyruvate, and vice versa.

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23-34
E. Isoenzyme
• Example: lactate dehydrogenase (LDH)
• The enzyme has four subunits (a tetramer of H and M
chains).
• H4 is present predominately in heart muscle.
• M4 is present predominantly in the liver and in skeletal
muscle.
• H3M, H2M2, and HM3 also exist.
• H4 is allosterically inhibited by high levels of pyruvate (its
product) and has a higher affinity for lactate (its substrate) than
does the M4 enzyme, which is optimized for the opposite
reaction. The M4 isozyme favors the production of lactate.
• H4 in serum correlates with the severity of heart attack.
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23-35
E. Isoenzyme
• Example: lactate dehydrogenase (LDH)

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23-36
23 Enzymes in Medicine
• Most enzymes are confined within the cells of the body.
However, small amounts of them can also be found in body
fluids such as blood, urine, and cerebrospinal fluid. The level
of enzyme activity in these fluids can easily be monitored. This
information can prove extremely useful: Abnormal activity
(either high or low) of particular enzymes in various body fluids
signals either the onset of certain diseases or their
progression.

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23-37
• Enzyme assays useful in medical diagnosis and their
activities in normal body fluids.

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23-38
• For example, a number of enzymes are
assayed (measured) during myocardial
infarction to diagnose the severity of the
heart attack. Dead heart muscle cells spill
their enzyme contents into the serum. As a
consequence, the level of creatine
phosphokinase (CPK) in the serum rises
rapidly, reaching a maximum within two
days. This increase is followed by a rise in
aspartate aminotransferase (AST; formerly
called glutamate-oxaloacetate
transaminase, or GOT). This second
enzyme reaches a maximum two to three
days after the heart attack. In addition to
CPK and AST, lactate dehydrogenase
(LDH) levels are monitored; they peak after
five to six days.
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23-39
• In infectious hepatitis (disease of the liver), the alanine
aminotransferase (ALT; formerly called glutamate-pyruvate
transaminase, or GPT) level in the serum can rise to 10 times
normal. There is also a concurrent increase in AST activity in
the serum.

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23-40
• In some cases, administration of
an enzyme is part of therapy.
After duodenal or stomach ulcer
operations, for instance, patients
are advised to take tablets
containing digestive enzymes that
are in short supply in the stomach
after surgery. Such enzyme
preparations contain lipases, either
alone or combined with proteolytic
enzymes. They are sold under
such names as pancreatin, Arco-
lase, and Ku-zyme.
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23-41
23 Transition State Analogs
• An enzyme lowers the activation energy for a reaction, making
the transition state more favorable. It does so by having an
active site that actually fits best to the transition state
conformation rather than to the substrates or the products.
This has been documented by the use of transition-state
analog.
Pyrrole-2-carboxylate
mimics the planar transition
state of the reaction.

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23-42
• Transition-state analogs have been used with many
enzymes to help verify a suspected mechanism and
structure of the transition state, as well as to inhibit an
enzyme selectively. They are currently being used for
drug design when a specific enzyme is the cause of a
disease.
• In 1969, William Jencks proposed that an immunogen (a molecule
that elicits an antibody response) would elicit antibodies with
catalytic activity if the immunogen mimicked the transition state
properties (shape, charge) of the reaction. Richard Lerner and Peter
Schultz, who created the first catalytic antibodies, verified this
hypothesis in 1986. Because an antibody is a protein designed to
bind to specific molecules on the immunogen, the antibody will, in
essence, serve as a fake active site.
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23-43
• Absyme: an antibody that has catalytic activity because
it was created using a transition state analog as an
immunogen.
The reaction of pyridoxal phosphate and an amino

acid to form the corresponding 𝛂-ketoacid and
pyridoxamine phosphate is a very important reaction
in amino acid metabolism.
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23-44
23
End
Chapter 23
Enzymes
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Lecture 7.1-Enzymes

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23

© 2006 Thomson Learning, Inc.

© 2006 Thomson Learning, Inc.

5. Isomerase: CH2 OPO 3 2 - CH2 OPO 3 2 -

© 2006 Thomson Learning, Inc.

In any case, an apoenzyme

© 2006 Thomson Learning, Inc.

• Competitive inhibitor: any substance that binds to the

© 2006 Thomson Learning, Inc.

© 2006 Thomson Learning, Inc.

Both the lock-and-key and induced-

© 2006 Thomson Learning, Inc.

Many cases of noncompetitive inhibition

© 2006 Thomson Learning, Inc.

If we compare enzyme activity

© 2006 Thomson Learning, Inc.

If the inhibitor is noncompetitive, it cannot be displaced by addition of excess substrate

© 2006 Thomson Learning, Inc.

© 2006 Thomson Learning, Inc.

© 2006 Thomson Learning, Inc.

The final product in the chain may inhibit the activity of

• An example is trypsin, a digestive enzyme.

© 2006 Thomson Learning, Inc.

© 2006 Thomson Learning, Inc.

© 2006 Thomson Learning, Inc.

• Example: lactate dehydrogenase (LDH) catalyzes the

© 2006 Thomson Learning, Inc.

© 2006 Thomson Learning, Inc.

© 2006 Thomson Learning, Inc.

© 2006 Thomson Learning, Inc.

© 2006 Thomson Learning, Inc.

© 2006 Thomson Learning, Inc.

The reaction of pyridoxal phosphate and an amino

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