Download Section 5: Enzymes, Equilibrium, Energy and the

Small World Initiative Instructor Guide Section 5: Enzymes, Equilibrium, Energy and the Metabolic Inhibitors Section 5: Enzymes, Equilibrium, Energy and the
Metabolic Inhibitors
TOPICS
Enzyme kinetics
Binding equilibrium
Free energy
Metabolic reactions/energy production
Molecular binding specificity
Mechanism of action of metabolic inhibitors
SUMMARY
We continue our investigation of antibiotic specificity by introducing the metabolic
inhibitors. The two different classes of metabolic inhibitors use two different mechanisms
to achieve specificity. Like the β-lactams, the sulfonamides achieve specificity by
binding to and inhibiting a target molecule not present in eukaryotes. In contrast,
trimethoprim is representative of those antibiotics that target a specific region of a
molecule present in both prokaryotes and eukaryotes. The subtle structural difference
between the prokaryotic and eukaryotic version of the target is such that binding affinity
of the antibiotic to the eukaryotic version is reduced relative to the prokaryotic version.
We use the metabolic inhibitors as a context for discussing enzyme kinetics and energy.
Instructors may also wish to include the key conserved metabolic pathways (glycolysis,
tricarboxylic acid cycle, oxidative phosphorylation) as well as highlighting the metabolic
diversity of the prokaryotes, although that content is not represented in the slides.
LEARNING GOALS
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Be able to explain the mechanism of action of metabolic inhibitors at the
molecular level
Know the factors that determine rates of chemical reactions.
Know the factors that determine equilibrium of chemical reactions.
Explain how enzymes achieve substrate specificity
Describe the molecular basis for antibiotic-target “binding” (covalent, noncovalent bond formation)
Explain binding equilibrium as it pertains to antibiotic-target binding (and how it
affects efficacy of an antibiotic)
Describe how metabolic inhibitors achieve prokaryotic specificity.
Additional metabolism-specific learning goals that could be incorporated
• Summarize the overarching goal of glycolysis, the tricarboxylic acid cycle and the
electron transport chain.
• Explain the electron transport chain and how cells extract energy from it.
• Know the role that oxygen plays in energy acquisition/catabolism
• Explain the mechanisms by which bacteria can survive in the absence of oxygen
Small World Initiative Instructor Guide •
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Section 5: Enzymes, Equilibrium, Energy and the Metabolic Inhibitors Know examples of terminal electron acceptors for aerobic respiration,
fermentation and anaerobic respiration.
Know the cellular targets of non-selective killing agents such as cyanide, heat,
bleach, etc.).
Explain chemiosmosis and its role in energy acquisition.
PRE-CLASS PREPARATION
Prior to class, students should read about thermodynamics of chemical reactions,
enzymes, and the significance of Gibbs free energy in chemical reactions. If biochemical
reactions of catabolism will be discussed, students should also gain a working knowledge
glycolysis, cellular respiration, and the electron transport system prior to class.
PRE-CLASS ASSESSMENT
1. Refer to first “Active Learning” activity for this section. These questions would
serve as appropriate pre-class assessment that could then be reviewed in an
electronic response-style during class.
2. If an enzyme contains a relatively non-polar active site, how is insertion of
charged amino acids at that site likely to affect its activity?
3. An antibiotic that affects activity of an intracellular enzyme must be able to cross
the cell membrane. Based on this, indicate structural features that you might
expect to be shared by antibiotics.
— Generally they are small molecules with varying polarity. Some antibiotics
gain access through pores and others cross the membrane itself. Most
antibiotics are generally non-polar, relative to water.
GUIDE TO THE POWERPOINT SLIDES
Outline
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Introduction to metabolic inhibitors
Chemical reactions
— Equilibrium
— Gibbs free energy
— ATP
Enzymes
Function
Specificity (sulfonamide as example)
Enzyme inhibitors (trimethoprim as example)
Introduction to metabolic inhibitors
The sulfonamides and trimethoprim are examples of metabolic inhibitors, drugs that
inhibit metabolic pathways that are vital for the life of the microbe. Folic acid is
important for human health; derivatives are necessary for synthesis of DNA and amino
acids and deficiency during pregnancy can lead to neural tube defects. Humans must
ingest folic acid in their diet while bacteria must synthesize their own folic acid
derivatives. The slides highlight the similarities and differences in the pathway between
bacteria and humans.
Small World Initiative Instructor Guide Section 5: Enzymes, Equilibrium, Energy and the Metabolic Inhibitors Bacteria synthesize their own folic acid derivatives
Folic Acid is important for human health
Intake from diet
Folic acid
(vitamin B9)
Dihydropteroate
diphosphate
(DHPP)
•  Derivatives are necessary for
synthesis of DNA and amino
acids
•  Especially important for rapidly
dividing cells
•  Deficiency in pregnancy can
lead to neural tube defects
conversion
in liver
Dihydropteroic acid
Folic acid
(vitamin B9)
Thymidine
synthesis
tetrahydrofolic acid (THF)
p-aminobenzoic acid
(PABA)
Dihydropteroate
synthase
(DHPS)
Dihydrofolic acid (DHF)
Dihydrofolate
reductase
(DHFR)
+
conversion
in liver
Dihydrofolic acid (DHF)
Dihydrofolate
reductase
(DHFR)
Amino
acid
synthesis
tetrahydrofolic acid (THF)
Thymidine
synthesis
Amino
acid
synthesis
The dihydropteroate synthase (DHPS) enzyme is present in bacteria, not humans and is a
target of the sulfonamides. The dihydrofolate reductase (DHFR) enzyme is present in
both humans and bacteria, yet trimethoprim targets only the bacterial version. The human
version is structurally different enough from the bacterial version that trimethoprim binds
with affinity several thousand-fold less than to the bacterial DHFR.
Metabolic inhibitor antibiotics interfere with function of critical
enzymes in the pathway
Dihydropteroate
diphosphate
(DHPP)
Dihydropteroate
synthase
(DHPS)
p-aminobenzoic acid
(PABA)
+
X
sulfonamides
Dihydropteroic acid
Folic acid
(vitamin B9)
conversion
in liver
Dihydrofolic acid (DHF)
Dihydrofolate
reductase
trimethoprim
X
(DHFR)
tetrahydrofolic acid (THF)
Thymidine
synthesis
Amino acid
synthesis
Chemical reactions
At this point, the following key concepts can be introduced:
- Equilibrium
- Gibbs free energy
- ATP
Since these topics are standard concepts covered in all general biology texts, we leave it
to each instructor to determine the content and level of detail required.
Key points:
— Collision theory: probability of molecules encountering each other is influenced
by reactant concentration, temperature, etc.
— Equilibrium is affected by the concentration
of products and reactants as well as the
change in free energy
— Rate is affected by the concentration of
Key Principle in Biology and Chemistry
products and reactants as well as by
temperature
Equilibrium
— Introduction of ATP as the universal energy
Most things are in equilibrium between two states
An atom can bond with and dissociate from a carboxylic acid
currency
More likely to be bound at lower pH
Small World Initiative Instructor Guide Section 5: Enzymes, Equilibrium, Energy and the Metabolic Inhibitors At this point, students should be able to meet the learning goals:
ü Know the factors that determine rates of chemical reactions.
ü Know the factors that determine equilibrium of chemical reactions.
Enzymes
Enzymes increase the rate of reaction but
do not alter equilibrium or the change in
free energy. Most biological reactions have
spontaneous rates that are too low to
sustain life, and so we depend on enzymes
to drive most reactions in the cell.
Figure 5.2
Enzyme function relies on a specific
interaction between the enzyme and
substrate. We eluded to specificity of
molecular binding interactions starting in
Section 3 with our discussion of the β-lactams as cell wall inhibitors. Now that noncovalent bonding interactions have been introduced (Section 4), we can elaborate on the
concept of molecular binding interactions using enzyme-substrate binding as our context.
Non-covalent binding interactions between enzyme and substrate dictate specificity.
Again, this is an opportunity to point out how structure relates to function.
Active learning
Activity type: Electronic response
The following electronic response questions are intended to help students self-assess and
synthesize the information presented.
True or False:
Question: 1) The free energy released will be greater if the reaction is catalyzed by an
enzyme.
2) The rate of the reaction increases in the presence of an enzyme.
3) The activation energy required for a reaction to proceed decreased in the
presence of enzyme.
1) The free energy released will be greater if the reaction is catalyzed by an enzymeFalse.
Enzymes lower activation energy but do not alter the change in free
energy.
2) The rate of the reaction increases in the presence of an enzyme.True. Enzymes increase
the rate of a reaction, essentially by increasing the
association of reactants. Enzymes tether reactants so that they are in the proper
orientation and there is a higher effective concentation.
Small World Initiative Instructor Guide Section 5: Enzymes, Equilibrium, Energy and the Metabolic Inhibitors 3) The activation energy required for a reaction to proceed decreased in the presence of
enzyme.
True. Enzymes decrease the activation energy, thus increasing rate of
reaction.
Choose all that apply:
Equilibrium of a reaction is affected by:
A. Change in free energy between products and reactants
B. Temperature
C. Concentration of reactants
D. Activation energy required
E. Rate at which the reaction proceeds
F. Presence of enzyme
Answer: A and C
Rate of a reaction is affected by:
A. Change in free energy between products and reactants
B. Temperature
C. Concentration of reactants
D. Presence of active enzyme
Answer: B, C, D
Enzyme inhibitors
Inhibitors usually bind to the active site of an enzyme (competitive inhibition) or bind to
a site other than the active site, which can result in a conformational change that affects
the active site (allosteric inhibition). Inhibition of the enzyme blocks the reaction,
preventing the formation of reaction products.
Sulfonamide structure resembles that of PABA
Sulfonamides mimic structure of PABA, “tricking”
DHPS to catalyze the wrong reaction
Dihydropteroate
diphosphate
(DHPP)
Dihydropteroate
synthase
(DHPS)
+
• 
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Enzyme recognizes sulfonamide as PABA, adds it to DHPP
Downstream reactions fail
p-aminobenzoic acid
(PABA)
sulfonamides
Dihydropteroic acid
Wrong
product
created
Dihydropteroate
diphosphate
(DHPP)
p-aminobenzoic acid
(PABA)
Dihydropteroic acid
sulfonamides
p-aminobenzoic acid (PABA) is the natural substrate of DHPS; sulfonamides mimic the
structure of PABA, such that DHPS binds to PABA and catalyzes reaction with DHPP
leading to formation of the wrong product. Trimethoprim inhibits DHFR, an enzyme
downstream of DHPS, through competitive inhibition.
Small World Initiative Instructor Guide Section 5: Enzymes, Equilibrium, Energy and the Metabolic Inhibitors In both cases (sulfonamides and trimethoprim) it is
imperative that the binding affinity of the antibiotic is
strong enough to compete with binding of the normal
substrate. Trimethoprim’s affinity for bacterial DHFR
is several thousand times greater than its affinity for
human DHFR.
Binding affinity of antibiotic for substrate must be
great enough to compete with binding of the
normal substrate
At this point, students should be able to meet the
learning goals:
ü Explain how enzymes achieve substrate specificity
ü Describe the molecular basis for antibiotic-target “binding” (covalent, noncovalent bond formation)
ü Explain binding equilibrium as it pertains to antibiotic-target binding (and how it
affects efficacy of an antibiotic)
Active learning
Activity type: Think-Pair-Share
Question: 1) How do the sulfonamides achieve prokaryotic specificity?
2) How does trimethoprim achieve prokaryotic specificity?
Sulfonamides affect biochemical reaction not present in humans
Dihydropteroate
diphosphate
(DHPP)
Dihydropteroate
synthase
(DHPS)
+
p-aminobenzoic acid
(PABA)
sulfonamides
Dihydropteroic acid
Folic acid
(vitamin B9)
Bacteria-specific
The first question (how do the sulfonamides
achieve prokaryotic specificity) should be
relatively easy. Instructors can point out that, just
as with the β-lactamases, sulfonamides target
an enzyme not present in mammalian cells.
conversion
in liver
The second question (how does trimethoprim
achieve prokaryotic specificity) may not be as
Thymidine
synthesis
straight
tetrahydrofolic acid (THF)
Intake from diet
Amino acid
forward, but
synthesis
+
the answer sets the stage for future discussions of the
sulfonamides
protein synthesis inhibitors and further increases
student understanding of molecular structure as it
relates to function. In brief, trimethoprim targets an
enzyme (DHFR) present in human cells, but the
trimethoprim
X
human version of this enzyme is structurally
Intake from diet
different such that binding affinity of the drug to the
human version of DHFR is several thousand fold less than to the prokaryotic version of
DHFR .We will discuss similar examples in upcoming sections.
Dihydrofolic acid (DHF)
Dihydrofolate
reductase
trimethoprim
X
(DHFR)
Trimethoprim has much lower binding affinity for the human version of DHFR
Dihydropteroate
diphosphate
(DHPP)
Dihydropteroate
synthase
(DHPS)
human-specific
p-aminobenzoic acid
(PABA)
Dihydropteroic acid
Folic acid
(vitamin B9)
Bacteria-specific
conversion
in liver
Dihydrofolic acid (DHF)
Dihydrofolate
reductase
(DHFR)
tetrahydrofolic acid (THF)
human-specific
Thymidine
synthesis
Amino acid
synthesis
Small World Initiative Instructor Guide Section 5: Enzymes, Equilibrium, Energy and the Metabolic Inhibitors At this point, students should be able to meet the learning goals:
ü Be able to explain the mechanism of action of metabolic inhibitors at the
molecular level
ü Describe how metabolic inhibitors achieve prokaryotic specificity.
POST-CLASS ASSESSMENT
1. Predict the efficacy of trimethoprim if the concentration of normal substrate
(dihydrofolic acid, DHF) is increased.
The efficacy of trimethoprim will be decreased. With more of the normal substrate
present, it is more likely to bind to the target enzyme, decreasing the effect of
trimethoprim.
2. Compare and contrast the interaction of β-lactams with transpeptidase and that of
sulfonamides and their target (dihydropteroate synthase, DHPS). Consider the molecular
binding interactions as well as the relationship between the structures of the enzyme and
antibiotic. Specifically, indicate what is being inhibited (the enzyme or some downstream
reaction). Is the inhibition reversible or irreversible? Explain. Indicate how each
antibiotic achieves prokaryotic specificity.
Just as the β-lactam antibiotics mimic the structure of the normal substrate (Dala-D-ala) for transpeptidase, the sulfonamides also mimic the structure of the
normal substrate (PABA) for DHPS. In both cases, the enzyme binds to the
antibiotic as if it were the normal substrate. In the case of β-lactams, a covalent
bond is formed between the enzyme and the antibiotic, thus preventing
downstream reactions from ensuing. In the case of the sulfonamides, the enzyme
catalyzes covalent bond formation between the wrong products (DHPP and the
antibiotic instead of between DHPP and PABA). The downstream reactions are
prevented because the required intermediate product (dihydropteroic acid)
cannot be formed. The DHPS enzyme remains active and thus the action of the
antibiotic is reversible—as antibiotic is removed (as concentration decreases) the
normal reaction can proceed. In contrast, the action of β-lactams is irreversible
because the enzyme activity is irreversibly destroyed by nature of the covalent
bond formed between it and the antibiotic. Both achieve prokaryotic specificity by
acting on enzymes not present in mammalian cells.
3. How might trimethoprim concentration affect its ability to inhibit?
The binding equilibrium is affected by concentration. At lower concentration, less
will be in the “bound” state and therefore at lower concentrations it will be less
effective as a competitive inhibitor.
RESOURCES
Enzymes
http://www.youtube.com/watch?v=E-_r3omrnxw&feature=related
Dynamic nature of molecular binding
http://www.youtube.com/watch?v=8xQtaWEroWM&feature=youtu.be