Download Key Concepts

Key Concepts:
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Mechanism of HIV Infection of Helper T Cells
Enzymes as Biological Catalysts
 Enzymes Lower Activation Energy
 Enzyme-Substrate Complex
 Michaelis-Menten Kinetics
 Competitive vs. Noncompetitive Inhibitors
Reverse-Transcriptase Inhibitors (AZT)
 Synthesis of Viral DNA from RNA Template
 AZT as a Thymidine Analog
Protease Inhibitors
The Deadly HIV Virus
HIV (human immunodeficiency virus), the virus that causes AIDS, is one of the
hottest areas of medical research today. It is estimated that 30.6 million people
throughout the world were infected with HIV by the end of 1997. After a person has
been infected with HIV, the virus usually remains dormant for long periods of time.
Then the virus begins a cycle of attacking cells of the immune system by
incorporating its genetic material into the cells, using the immune cells' machinery to
make more viruses from the incorporated genetic material, and then breaking the cells
apart (killing them) so that the new viruses can infect more cells. In this manner, the
immune system is weakened, so that the body can no longer defend itself against the
pathogens that it encounters every day. Sadly, patients typically die within a few years
of showing symptoms of AIDS (i.e., signs of drastically decreased immunity due to
the virus's attack on the cells of the immune system). Because so many people are
affected by HIV, and because the virus is so deadly, much research has been devoted
to finding ways to fight this epidemic. Researchers are seeking treatments for HIVinfected individuals, cures, and vaccines. So far, the most promising findings have
been treatments to lessen the damage to the immune system from HIV; several
treatments that have been developed have dramatically improved the outlook for
many HIV patients. However, these treatments have many severe side effects, and in
most cases are too costly to be administered widely in many parts of the world. Thus,
more research is needed to improve the available treatments, making them more
tolerable to patients and more accessible, and to continue the search for a cure for
HIV and a vaccine, which would prevent the infection in the first place.
This tutorial describes two of the most successful treatment options available today:
reverse-transcriptase inhibitors (e.g., AZT) and protease inhibitors. These treatments
interfere with enzymes that are needed for HIV to make copies of itself, a key step in
the virus's attack on the cells of the immune system. In order to understand how these
treatments help, we must first discuss the immune cells that come under attack, and
the mechanism by which the virus kills these cells if left untreated.
HIV Attacks Helper T Cells
Our body's immune system contains many different types of cells, but only one of
these cell types, known as helper T cells, is attacked by HIV. Helper T cells are
necessary to stimulate the activation of other immune cells that attack infectious
particles (antigens) in the body. When these cells come under attack by HIV, the
immune system can no longer function effectively, and the body is incapable of
combating many types of foreign infections and cancers. A more detailed
description of normal immune function, and why helper T cells are critical to the
immune system, is given in the peach box, below.
The Immune System: The Body's Defense Against Infection
The immune system is a collection of cells found in the blood and in
tissues throughout the body that help to protect the body from infection.
These cells perform a variety of nonspecific and specific functions to
defend the body against harmful particles (e.g., bacteria, viruses, toxins,
and cancerous cells). Nonspecific responses do not require that the cell
recognize a particular type of infectious agent; such responses include
inflammation (which destroys or inactivates invading particles and
prepares a site for tissue repair) and phagocytosis (which engulfs the
harmful particles using large, specialized immune cells). Specific
immune responses are directed against a particular infectious agent, and
involve the recognition and attack of particular particles, known as
antigens.
Types of Immune Cells
The most numerous cells of the immune system are the leukocytes
(white blood cells), which are produced in the bone marrow and travel
through the blood to other tissues, where they do their infection-fighting
work. Other immune cells include plasma cells (which make and secrete
antibodies), macrophages (large cells that engulf invading particles),
and mast cells (which secrete locally-acting chemicals involved in
inflammation). The classification of the cells of the immune system is
summarized in Figure 1, below. Because the leukocytes are the most
numerous immune cells, and also include the type of cells attacked by
HIV, we shall focus on the different types of leukocytes, and how they
protect the body from infections.
Figure 1
This diagram summarizes the classification and
functions of the major types of cells in the immune
system. Note that the helper T cells, a specific type of
leukocyte, are the target for HIV infection.
There are five major classes of leukocytes: neutrophils, basophils,
eosinophils, monocytes, and lymphocytes. The neutrophils, basophils,
eosinophils, and monocytes, like many of the other immune-cell types
described above, participate in nonspecific immune defenses, including
stimulating inflammation, engulfing particles, and making the body
more sensitive to invading particles. The lymphocytes, which is the
class of leukocytes that the HIV virus targets, are a far more complex
class of leukocytes that participate in specific immune defenses, i.e.,
they must recognize the specific material being attacked. A particle that
triggers a specific immune response, such as a toxic molecule or a
special protein bound to the outside of a bacterium or infected cell of the
body, is known as an antigen.
Lymphocytes' Attack on Antigens
How do the lymphocytes recognize and attack antigens in the body?
First, a lymphocyte must encounter and recognize the antigen. Each
lymphocyte in the body has a receptor (Figure 2) that can bind to a
specific antigen; the body contains millions of lymphocytes with
different receptors to recognize a huge number of different antigens that
the body might encounter. The receptors are proteins with a specific
amino acid sequence at the binding site, giving the binding site a shape
that will allow it to bind to a specific antigen (depending on the shape
and polarity of the antigen). When a lymphocyte encounters the antigen
for which it has a receptor, the receptor binds to the antigen.
Figure 2
This figure shows how the receptor on a lymphocyte
recognizes and binds to a specific antigen. Here, a
cytotoxic T cell (turquoise) recognizes a cell that has
been infected by a virus (mauve), because the T cell's
receptor binds to a viral protein (yellow) on the
outside of the cell. The particle that binds to the
receptor (i.e., the viral protein on the outside of the
cell) is the antigen.
Once a lymphoctye has bound to an antigen, the lymphocyte then
becomes "activated". The lymphocyte undergoes a series of cell
divisions to produce many cells that are identical to the one that first
recognized the antigen. Finally, the activated lymphocytes attack the
antigen, and all antigens of the same kind that are found throughout the
body.
To understand the activation and attack processes, we must differentiate
between the different types of lymphocytes. The principal types of
lymphocytes are called B cells, cytotoxic T cells, helper T cells, and
NK cells. (The designation "B" and "T" refer to where the cells mature.
Although all lympocytes originate in the bone marrow, the T cells then
travel to the thymus ("T" for "thymus") to mature; B cells mature in the
bone marrow ("B" for "bone"); NK stands for "natural killer", and it is
uncertain where these cells mature.) B cells recognize foreign antigens,
such as bacteria, viruses, and toxins. Cytotoxic T cells, on the other
hand, recognize as antigens the body's own cells that have become
cancerous or infected by a virus.
B cells, cytotoxic T cells, and helper T cells all contain receptors that
bind to antigens and become activated. Once activated, helper T cells do
not attack the antigen themselves, but help to activate B cells and
cytotoxic T cells by secreting special chemical signals. With very few
exceptions, these signals from helper T cells are required for the
activation of the B cells and cytotoxic T cells. Helper T cells are the
cells in the body that are attacked by HIV. Hence, HIV disrupts the
immune system by destroying the cells needed for B cells and
cytotoxic T cells to become activated and thus attack antigens in the
body.
With the chemical signal from helper T cells, the B cells and cytotoxic T
cells that have recognized and bound to an antigen can then become
activated and begin their attack. The two types of lymphocytes differ in
the types of antigens they recognize and attack, and in their mode of
attack. B cells, which recognize foreign antigens, do not attack the
antigen directly. Instead, they release particles known as antibodies,
which guide other cells (e.g., macrophages and NK cells) to the antigen
to engulf or neutralize the antigen. Cytotoxic T cells, which recognize
the body's own cells that are cancerous or infected as antigens, directly
attack their antigens, killing the cancerous or infected cells.
Overview of HIV's Attack on the Immune System
How does HIV invade and kill helper T cells, thereby depleting our immune system?
To answer this question, we must first clarify what we mean by the term virus. Then
we shall outline the major steps in the life cycle of HIV, and how these steps lead to
the destruction of helper T cells. As you read this description, note that many of these
steps are made possible by particular enzymes, biological catalysts that change the
mechanism (and therefore the kinetics) of a biochemical reaction in order to enable
the reaction to proceed with a smaller activation energy. (A more in-depth explanation
of how enzymes work is given in the section below, "Enzymes as Biological
Catalysts".)
A virus is often classified as a living thing, although it is not made up of cells, like
other organisms. Viruses consist of proteins surrounding genetic material.
Depending on the virus, this genetic material may be either DNA (the form in which
our cells' chromosomes contain genetic material) or RNA (the form used for the
expression of genetic information in cells, and the form in which some viruses carry
their genetic information). Of the two types of viruses, DNA viruses and RNA
viruses, HIV represents the second. A virus containing RNA is known as a
retrovirus. In order to reproduce, a retrovirus must attach to a cell of the infected
organism, insert its RNA into the cell, and make a DNA copy of the RNA. This DNA
copy then incorporates into the cell's own chromosomes (which are made of DNA),
and uses the cell's biochemical machinery to replicate the viral DNA (along with the
host cell's DNA), make viral proteins from the DNA that is replicated, and assemble
new viral particles from these proteins.
The steps by which HIV infects and kills helper T cells are described below, and can
be viewed in Figure 3.
Figure 3
This schematic shows the major steps in HIV's attack on the human
immune system. The numbers correspond to the numbered steps in the
description of the infection cycle, below.
HIV Infection Cycle
1. The first step in HIV's attack on helper T cells is attaching to the cell. Helper T
cells contain proteins called CD4 proteins in their cell membrane that extend
outside of the cell. Normally, these proteins help the cells to bind to antigens
(infectious particles) in order to stimulate activation of the helper T cells, and
2.
3.
4.
5.
6.
7.
8.
9.
they are also required for normal T cell development. Unfortunately,
however, CD4 proteins also function as receptors for HIV, allowing the
virus to attach itself to the cell and thereby gain access to the cell's
biochemical machinery.
Once the virus has attached to a helper T cell, it injects its genetic
information (as RNA) into the cell, along with the enzyme reverse
transcriptase.
Reverse transcriptase catalyzes the production of DNA from the viral
RNA, making a DNA copy of the virus's genetic material. This DNA copy
is capable of incorporating itself into the cell's genetic material, because it is
now in the same form as the cell's chromosomes. Hence, the step catalyzed
by reverse transcriptase is one of the most important steps in the infection
cycle.
The viral DNA copy then enters the nucleus of the infected helper T cell,
where it is incorporated into the cell's genetic material (i.e., the
chromosomes).
Using the cell's own DNA-replication mechanisms, the viral DNA
replicates.
Using the cell's mechanisms for producing proteins from the genetic
information contained in DNA, many copies of the proteins needed by the
virus are made from the replicated HIV DNA. As part of this step, RNA
copies of the viral DNA are made.
When they are first synthesized, the proteins are too long (containing extra
fragments) to be assembled into new viruses. They must be cut to their
proper size. The HIV enzyme protease, which is produced by the cell's
biochemical machinery from the viral DNA incorporated into the cell's
chromosomes, catalyzes the cutting of these proteins to their proper size.
New HIV particles (viruses) are assembled inside the cell from the cut viral
proteins and the viral RNA copies.
Once assembled, the new viruses then burst out of the host cell (killing it)
and invade new cells, continuing the infection.
As you can see from the steps outlined above, enzymes play a vital role in HIV's
attack on helper T cells. The generation of a DNA copy of the viral genome by
reverse transcriptase (Step 3) and the cleavage of viral proteins (Step 7) by protease
are two important processes catalyzed by enzymes. Therefore, the enzymes reverse
transcriptase and protease are major target sites for HIV-fighting drugs. Before
we can explain how these drug treatments work to combat HIV, we must first discuss
how enzymes work to catalyze biochemical reactions.
Questions on HIV's Attack on the Immune System
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Briefly, explain why HIV must infect a host cell in order to reproduce.
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Reverse transcriptase is injected by HIV into the host cell, along with the viral
RNA. All of the other proteins needed by HIV are not injected into the host
cell, but are synthesized inside the cell. Briefly, explain why reverse
transcriptase must be injected, rather than synthesized inside the cell.
Enzymes as Biological Catalysts
Many biological reactions, such as most normal reactions of the cells, and the
reactions employed by HIV to replicate itself, are thermodynamically favorable
(G<0) or can be made thermodynamically favorable by coupling with other
reactions. Recall that thermodynamics tells us whether a reaction (or process) is
spontaneous under a specified set of conditions. However, thermodynamics does not
tell us how fast (or the rate with which) a reaction will proceed. Rates of reactions are
the realm of chemical kinetics and are determined experimentally. From Arrhenius
Theory, the rate of a reaction is dependent on the activation energy (Ea), which is the
minimum amount of energy needed for a reaction to occur (See Figure 4, Path A).
Hence, even though a reaction is thermodynamically favorable, it still can not occur
unless there is enough energy available (an amount greater than or equal to the
activation energy) to initiate the reaction. If sufficient activation energy cannot be
supplied (i.e., the rate of the reaction is too slow), then a catalyst may be used. Recall
from the introduction to the Experiment that a catalyst is a substance that increases the
rate of a reaction without being consumed. A catalyst influences the mechanism
(pathway) of a reaction, but does not affect the thermodynamics (G). A catalyst
allows the reaction to proceed by an alternate mechanism that has a lower activation
barrier (Figure 4, Path B). Often biological reactions require catalysts called enzymes
to change the pathway (mechanism) of the reaction, and thus lower the activation
energy. When a catalyst changes the reaction pathway to lower the activation energy,
the reaction rate is increased, but the thermodynamics of the reaction are not
changed. That is, a catalyst cannot form products that are not allowed by
thermodynamics (G); it does increase the rate of forming the products that are
allowed by thermodynamics. Enzymes are molecular catalysts in biological
systems, and are usually proteins. Virtually all biological reactions are catalyzed by
enzymes.
Figure 4
The schematic on the left, Path A, (blue) shows the high activation energy
associated with an uncatalyzed reaction, and the schematic on the right,
Path B, (red) shows the lower activation energy associated with the same
reaction in the presence of a catalyst.
How Enzymes Work
Enzymes are very specific to the substrates (reactants) and reactions that they can
catalyze. Enzymes work by binding the substrate into a favorable orientation in an
enzyme-substrate complex (an intermediate in the reaction), which promotes the
making and breaking of chemical bonds. The substrate is bound to the active site (a
specific region of the enzyme), is converted to the product, and then released from the
active site. The active site is a three-dimensional crevice, and is usually only a small
portion of the total volume of the enzyme (Figure 5).
Figure 5
This is a schematic diagram showing the
active site of an enzyme (green)
The substrate is "bound" to amino acids in the active site of the enzyme by multiple
intermolecular interactions, including charge-charge interactions (electrostatics),
hydrogen bonding, and van der Waals forces. Enzymes are often very sensitive to
changes in pH and temperature, in part because these changes can affect the shape and
charge of the active site, thus changing the interaction with a substrate. If the optimal
conformation of the enzyme is lost, the enzyme becomes nonfunctional (i.e., the
substrate can no longer bind to active site).
Enzyme Kinetics
The study of the kinetics of a reaction that is catalyzed by an enzyme is essentially
like the kinetics studies that you perform in lab, but involves a few additional,
specialized concepts. The model for understanding the kinetic properties of most
enzymes is known as the Michaelis-Menten model. This model proposes the
following mechanism for enzyme catalysis. First, the enzyme (E) and substrate (S)
come together to form an enzyme-substrate complex (ES), as shown in Equation 1,
below. The reaction occurs, and the substrate is converted to the product of the
reaction. Then, the enzyme-substrate complex is broken apart, yielding enzyme (E)
plus product (P), as shown in Equation 2, below.
Mechanism:
(1)
(2)
Overall reaction:
(3)
The Michaelis-Menten model assumes that only a negligible amount of enzymesubstrate complex reverts to reactants(i.e., k-1 << k1 in Equation 1). The rate of
formation of product (shown below in Equation 4) can be determined from Equation 2
in the mechanism written above
Rate of formation of Product = k2[ES]
(4)
and the rate of formation of the intermediate ES (shown in Equation 5) can be
determined from Equations 1 and 2 in the mechanism written above
Rate of formation of ES = k1[E][S] - (k2 + k-1)[ES]
(5)
Using the steady-state approximation (i.e., the assumption that the concentrations of
intermediates (in this example, ES) stay constant while the concentrations of reactants
and products change), and making several substitutions (which are not shown here),
we can form an equation for the rate of formation of the product (Equation 6).
(6)
where [E]o is the initial concentration of free enzyme, [S] is the substrate
concentration, and Km is a constant specific to a given enzyme known as the
Michaelis-Menten constant. The value of Km relates to the rate constants shown in
Equations 1 and 2, as given by Equation 7:
(7)
The Michaelis-Menten constant (Km) is very important, because it can be determined
experimentally and describes the catalytic power of an enzyme. Km can also be used
to predict the rate of a reaction catalyzed by an enzyme, given the starting conditions.
Enzyme Inhibition
Enzyme function may be hampered by the addition of molecules or ions called
inhibitors. Many drugs work by inhibiting (slowing or stopping) various enzymes.
Inhibitors function by forming an enzyme-inhibitor complex, which impedes the
ability of the enzyme to convert substrate to product.
Inhibitors can be reversible or irreversible. Irreversible inhibitors are tightly bound to
the enzyme, often through covalent bonds. Hence, the enzyme-inhibitor complex
does not dissociate (or it dissociates very slowly). Reversible inhibitors bind through
electrostatic interactions (e.g., dipole-dipole interactions). These weaker interactions
form an enzyme-inhibitor complex that dissociates very quickly. Reversible inhibitors
are generally classified as competitive or noncompetitive. (Irreversible inhibitors do
not dissociate; therefore, they cannot be classified as competitive or noncompetitive.)
A competitive inhibitor often binds to an enzyme's active site (i.e., competes with
the normal substrate for the enzyme's active site), thus preventing substrate molecules
from binding to the enzyme and reacting. Note that both the substrate and the
inhibitor bind loosely enough to the enzyme that the enzyme-substrate and enzymeinhibitor complex dissociate rapidly. In a typical scenario, a single molecule of
enzyme collides with a substrate molecule and binds it for a short time. Either the
substrate is rapidly converted to product, or it quickly "falls off" the enzyme
unchanged. The enzyme will then collide with and bind another molecule, perhaps an
inhibitor this time. While the inhibitor is bound, the enzyme does not convert any
substrate to product, because the competitive inhibitor and substrate do not bind
simultaneously. After a short period, the inhibitor dissociates, and another collision
between the enzyme and either substrate or inhibitor results in a new binding
event. Note that competitive inhibitors prevent the substrate from binding.
Noncompetitive inhibitors usually bind to a different site on the enzyme (i.e., not the
enzyme's active site for the normal substrate). When a noncompetitive inhibitor binds
to an enzyme, the shape of the enzyme may be changed. This changes the mechanism
of the enzyme reaction and slows the rate at which the substrate is converted to
product. Note that the presence of a noncompetitive inhibitor does not completely
prevent the substrate from binding, it just slows down the rate at which the substrate is
converted to product.
The most common method of determining whether a reversible inhibitor is
competitive or noncompetitive is to see what effect the inhibitor has on the rate at
which product is formed using different concentrations of the substrate. Recall, the
enzyme randomly binds either a competitive inhibitor or substrate molecule, but not
both, depending on which it collides with first. A competitive inhibitor will be more
effective at low substrate concentrations because the enzyme is more likely to collide
with and bind the inhibitor if there are fewer substrate molecules in the vicinity. A
noncompetitive inhibitor will be equally effective at low and high substrate
concentrations. Recall, noncompetitive inhibitors bind along with substrate, not
instead of it. Hence, adding more substrate does not influence whether the enzymeinhibitor complex forms. In other words, to determine whether an inhibitor is
competitive or noncompetitive, one compares the measured value of Km in the
presence and absence of inhibitor. For a competitive inhibitor, the measured value of
Km increases in the presence of inhibitor. For a noncompetitive inhibitor, the
measured value of Km remains the same. As will be discussed below, the most
successful treatments for HIV have resulted from using inhibitors for the two enzymes
reverse-transcriptase and protease.
Questions on Enzymes as Biological Catalysts

A researcher has isolated an enzyme and found its active site to be lined with
polar amino acids. From other biological studies, the researcher knows that
this enzyme must be used by the cell to catalyze the breakdown either of
hydrophobic toxins (like DDT) or of ascorbic acid, a water-soluble vitamin.
Using the researcher's new discovery about the active site of the enzyme,
predict which is the actual substrate (hydrophobic toxins or ascorbic acid) for
the enzyme. Briefly, explain your reasoning.

The Michaelis-Menten constant (Km) can be used as an index of the stability
of the enzyme-substrate complex. Does a high Michaelis-Menten constant
indicate a stable or an unstable enzyme-substrate complex? Briefly, explain
your reasoning.

A student suggests that one way to overcome the effect of an enzyme inhibitor
would be to add a large amount of substrate to the reaction vessel (i.e., saturate
the reaction with substrate).
a. Can a competitive inhibitor be overcome by saturating the reaction with
substrate? Briefly, explain your answer.
b. Can a noncompetitive inhibitor be overcome by saturating the reaction with
substrate? Briefly, explain your answer.

How does an enzyme affect the equilibrium of a reaction?
Enzyme-Targeting Drugs to Fight HIV
We have described the role of two critical enzymes, reverse transcriptase and
protease, in HIV's ability to infect helper T cells, make copies of itself, and ultimately
destroy the cells, depleting the body's immune system. We have also seen how
enzymes catalyze biochemical reactions. Now, we turn our focus to two of the most
successful treatments for HIV, reverse-transcriptase inhibitors and protease inhibitors.
As their names imply, these drugs inhibit enzymes that are necessary for HIV to
replicate itself inside helper T cells and deplete the body's immune system.
Reverse-Transcriptase Inhibitors: AZT
Recall from Figure 3, above, that reverse transcriptase catalyzes the formation of a
DNA copy of the virus's RNA genetic information. Without the reverse transcriptase
enzyme, the virus HIV cannot make DNA copies of its genetic material (i.e., the
RNA), because the activation energy for the uncatalyzed reaction is too large. The
DNA copy is essential for the virus to take over the infected cell's machinery and
produce new copies of itself. Therefore, reverse transcriptase has long been an
obvious target in the scientific battle against HIV and AIDS.
How does reverse transcriptase lower the activation energy to enable the virus to
make the DNA copy of its RNA? The reverse-transcriptase enzyme (Figure 6) is a
protein consisting of two peptide subunits (amino-acid chains). At the interface of the
two subunits is an active site where a strand of viral RNA, together with the newlyemerging strand of DNA formed from the RNA template, can fit. A portion of the
larger subunit functions as a ribonuclease, digesting the RNA once the DNA copy has
been made. Except for the ribonuclease region, the two subunits are identical.
Figure 6
This is a three-dimensional CPK (space-filled)
representation of HIV Reverse Transcriptase,
complexed with the viral RNA (yellow) and the
newly-forming strand of DNA (pink). The enzyme
consists of two subunits, known as p51 (green) and
p66 (blue). A portion of the p66 subunit functions as
a ribonuclease and is shown in red.
Note: The coordinates for the model were
determined from x-ray crystallographic data, and the
image was rendered using SwissPDB Viewer and
POV-Ray (see References).
Note: To view the
reverse-transcriptase
enzyme interactively,
please use RASMOL, and
click on the button to the
left.
To more fully understand how reverse transcriptase enables the virus to make DNA
copies of its RNA, we must know something about the structure of DNA, and the
mechanism by which a strand of DNA is generated. The concepts are described in the
blue box, below.
Structure of DNA
DNA and RNA, like proteins, consist of chains of smaller buildingblock molecules. The building blocks for DNA and RNA are called
nucleotides (Figure 7). There are four different nucleotides in DNA, and
the sequence of these nucleotides in a DNA strand determines the
sequence of amino acids in the protein that will be made from the
genetic information contained in the DNA. RNA consists of a single
strand of nucleotides, and DNA consists of two strands of nucleotides
that are connected by hydrogen bonds along the length of the chain.
Each nucleotide has a complementary nucleotide with which it is paired
in the opposite chain. For instance, the nucleotide adenine is always
situated across from a thymidine nucleotide in a double-stranded piece
of DNA. As shown in Figure 7, each nucleotide consists of a single- or
double-ring structure (known as a pyrimidine or a purine, respectively),
attached to a sugar molecule. The sugar molecule contains two -OH
groups, known as the 5' -OH group and the 3' -OH group. At the 5' -OH
group, a phosphate (PO43-) group is attached to complete the nucleotide.
Figure 7
This figure shows the important features of a
nucleotide: the pyrimidine or purine (blue), the sugar
(black), and the phosphate group (green). The
pyrimidine or purine determines the identity of the
nucleotide. The oxygens in the 3' and 5' OH groups
are shown in red. The hydrogen from the 5' -OH
group is removed in order to form the bond with the
phosphate group.
Making a DNA Copy of RNA Genetic Material
To make DNA from RNA, a short piece of RNA, known as a primer,
that contains the proper sequence of amino acids to form complementary
pairs with a segment of the RNA chain to be copied (the "template"), is
aligned opposite the RNA strand to be copied and forms hydrogen bonds
with it. The 3' -OH group of the primer can now form a covalent bond
with the phosphate group of another nucleotide that complements the
next nucleotide on the template RNA strand. The new nucleotide will
hydrogen bond with the complementary nucleotide on the template
strand, as shown in Figure 8. Additional nucleotides are added in the
same manner, until the strand is complete.
Figure 8
This figure shows a strand of DNA being
synthesized, using an RNA strand (orange) as the
template. The part of the DNA strand that has
already been synthesized is at the bottom of the
right-hand side of the image. The hydrogen bonds
that form between complementary purines and
pyrimidines are shown as dotted magenta lines. A
new nucleotide (pink background) is added by
forming a covalent bond between the 3' -OH
group (red) of the last nucleotide on the existing
strand, and the phosphate group (green) of the
new nucleotide. The site of this bond is shown
with yellow arrows. Note that the new nucleotide
actually has three phosphate groups attached at its
5' -OH position; two of these will be removed
when the bond between the phosphate and the
existing strand is formed.
How does reverse transcriptase catalyze the formation of a DNA copy of HIV's RNA
genetic material (the reaction described in the blue box, above)? To synthesize DNA,
reverse transcriptase first positions the RNA strand (called the "template") into the
active site, together with an additional, special piece of viral RNA that serves as the
primer, or first piece, of the new strand. The primer RNA lines up opposite the
template RNA strand, forming hydrogen bonds between the complementary purines
and pyrimidines, as shown in Figure 8, above. Then, reverse transcriptase positions a
new nucleotide such that a covalent bond can form between the 3' -OH group of the
last nucleotide in the primer and the phosphate group of the new nucleotide (see
Figures 7 and 8). Reverse transcriptase lowers the activation energy for this
reaction by bringing the molecules in close proximity to one another in the active
site.
It is possible to inhibit the action of reverse transcriptase using drugs. Currently, six
drugs that act as inhibitors of reverse transcriptase are on the market for treating HIVinfected patients. Zidovudine, or AZT, is one of the earliest and best known of these
drugs; it is marketed under the brand name Retrovir. AZT (Figure 9) functions as an
analog for thymidine (Figure 10), one of the nucleotide building blocks of DNA. This
means that AZT has the same shape as thymidine, and therefore it can be
incorporated into the developing nucleic acid in place of a thymidine molecule.
The phosphate group attached to thymidine or AZT forms a bond with the 3' -OH
group of the preceding nucleotide in the developing DNA chain. When thymidine is
incorporated into the DNA chain, its 3' -OH becomes the binding site for the next
nucleotide's phosphate group. However, AZT lacks the -OH functional group that is
necessary to form a bond with the next nucleotide; in its place is an azido (-N3) group.
Because the azido group cannot form a bond with a phosphate group, no
additional nucleotides can be added once AZT is incorporated into the DNA
chain. Hence, reverse transcription stops after AZT is incorporated.
Figure 9
This is a two-dimensional
representation of AZT with a
phosphate group attached to the 5' OH group (i.e., the -OH group
attached to the 5' carbon of the
sugar). AZT stands for
"azidothymidine" because it
resembles thymidine but has an
azido (-N3) group in the 3' position
(i.e., attached to the 3' carbon) of the
sugar portion of the molecule. This
azido group terminates the nucleic
acid chain because it cannot bond to
another nucleotide.
Figure 10
The normal phosphorylated
thymidine molecule can be
phosphorylated (addition of a
phosphate group) to become one of
the nucleotide building blocks of a
DNA strand. The 3' -OH group (i.e.,
the -OH group attached to the 3'
carbon of the sugar) allows
thymidine to bond to another
nucleotide via the phosphate linkage,
continuing the nucleic acid chain.
A major problem with AZT is that the HIV virus quickly mutates, and strains that are
resistant to the drug may arise in patients who have been taking AZT for extended
periods of time. One strategy that doctors use to get around this problem is "multipledrug therapy." AZT is administered in combination with other reverse transcriptase
inhibitors or, increasingly, with one or more protease inhibitors (see next section
below). Thus, mutants that evolve with resistance to any one of the drugs are still
likely to be killed by the other drugs in the therapeutic regimen.
Protease Inhibitors: A New Line of Attack
In December 1995, the FDA approved a new type of drug for combating HIV. This
class of drug acts by inhibiting protease, the enzyme required by HIV to cut its protein
into the proper segments to assemble new viral particles. Protease inhibitors, used in
combination with two reverse transcriptase inhibitors, have proven to be quite
successful. In 80 to 90 percent of patients, this combination treatment reduces the
amount of HIV in the blood to an undetectable level.
As shown in Figure 3, the proteins produced from HIV's genetic material are larger
than the proteins needed to form new viral particles. In fact, a large "polyprotein" is
formed that contains several viral proteins joined together. The polyprotein must be
cleaved into the individual functional proteins. This cleavage is catalyzed by protease
(Figures 11 and 12). (Cells also contain proteases of their own; the protease described
in this section, however, refers to the protease that is made from HIV's genetic
information and is used to cleave HIV proteins.) This enzyme is a symmetric
homodimer, or a protein consisting of two identical peptide subunits. Like the active
site of reverse transcriptase, the protease active site lies at the interface of its two
subunits. The mechanism by which protease cleaves the HIV polyprotein will not be
discussed in this tutorial. Protease inhibitors reversibly bind to the protease
enzyme and, while bound, prevent the enzyme from cutting the viral protein
molecules down to their proper sizes.
Figure 11
This is a CPK representation of
protease with a protein substrate
(gold) occupying the active site.
The active site lies at the interface of
the two identical subunits (green and
purple).
Figure 12
This is a CPK representation of the
inhibited protease. The compound
DMP323 (blue), although not
approved for use in humans, is a
potent inhibitor of protease and is
used in scientific investigations to
Note: The coordinates for Figures 11
understand how protease inhibitors
and 12 were determined from x-ray
work, and how the virus might
crystallographic data, and the images
mutate to gain resistance to this class
were rendered using SwissPDB
of drugs.
Viewer and POV-Ray (see
References).
Note: To view the uninhibited
protease enzyme interactively,
please use RASMOL, and click on
the button above.
Note: To view the inhibited
protease enzyme interactively,
please use RASMOL, and click on
the button above.
Questions on Enzyme-Targeting Drugs to Fight HIV

AZT inhibits reverse transcriptase by acting as an analog for the nucleotide
thymidine.
a. Is AZT a competitive or a noncompetitive inhibitor?
b. Does AZT change the value of Km for reverse transcriptase?

The compound 4'-azidothymidine (ADRT) is another analog of thymidine. It
contains the 3' -OH group that AZT lacks, and therefore does not cause chain
termination in reverse transcriptase like AZT. However, ADRT has been
suggested as a possible competitive inhibitor of thymidine kinase, the
enzyme that adds the phosphate group to the 5' -OH of the sugar portion of
thymidine (or AZT) so that it can be incorporated into a DNA strand. The
reaction catalyzed by thymidine kinase is:
Thymidine Kinase + Substrate + Phosphate ---> Thymidine Kinase + SubstrateMonophosphate
where Substrate = Thymidine, ADRT, or AZT
Chen et al. found that the Km value for the thymidine kinase reaction with thymidine
as a substrate is 0.7 M, but the Km value for the thymidine kinase reaction with
ADRT as a substrate is 8.3 M. Using the definition of Km (Equation 7), together with
your understanding of competitive enzyme inhibition, predict whether ADRT will be
a good inhibitor or a poor inhibitor of thymidine kinase. Briefly, explain your
reasoning.

Based on your understanding of how AZT inhibits reverse transcriptase,
would you expect this drug to have any effect on normal cell function, as
well? Briefly, explain your answer.

Look at the three-dimensional representations of the protease molecule
complexed with a normal protein substrate (Figure 11) and with the inhibitor
DMP323 (Figure 12). Using your knowledge about inhibitors, and ignoring
minor differences in orientation between the two images, tell whether
DMP323 is a competitive or a noncompetitive inhibitor for protease. In one
short sentence, explain your reasoning.

Briefly, explain why AZT does not act as a protease inhibitor, using your
knowledge of enzymes and inhibitors.
Conclusion
To reproduce, HIV infects an immune cell called the helper T cell. (T cells help
control the body's response to many types of infections.) The HIV then uses the
machinery of the helper T cell to make copies of the HIV virus. The major
reproductive steps in HIV's infection of the helper T cell are (1) attaching to the cell,
(2) injecting RNA into the cell, (3) making a DNA copy of the genetic information
contained in HIV's RNA, (4) incorporating the viral DNA into the cell's
chromosomes, (5) replication of the viral DNA using the cell's machinery, (6)
producing proteins from the viral genetic information, using the cells machinery, (7)
cutting the proteins to proper size, (8) assembling new viral particles, and (9) bursting
out of the host cell to continue the infection.
As discussed in the tutorial, most of these steps are accomplished by using enzymes,
which, in biological systems, are proteins that catalyze reactions. Enzymes form an
enzyme-substrate complex with the normal reaction substrates. The formation of the
enzyme-substrate complex helps to lower the activation energy of the reaction. The
treatment to help HIV-infected people has been based on developing drugs that inhibit
these major reproductive steps. Inhibitors are drugs that work by forming an enzymeinhibitor complex, which impedes the ability of the enzyme-substrate complex to
form. The most successful treatments for HIV have resulted from using inhibitors for
the two enzymes reverse transcriptase (step 3 above) and protease (step 7 above).
These two types of inhibitors are described in the tutorial.
Since the discovery of HIV virus in 1984, much research has been conducted and
resulted in an increased understanding of the virus, and the development of drugs that
have been successful in the treatment (but not cure) of HIV. However, more research
is needed in order to effectively combat the global epidemic of HIV. Research is
continuing to develop new drug treatments for the disease. In addition to the inhibitors
described in the tutorial, current research is focusing on inhibitors that will block the
attachment of HIV to helper T cells (step 1 above), the integration of HIV's DNA into
the cell's genetic material (step 4 above), and the assembly of new viral particles (step
8 above). An inhibitor for the enzyme integrase (used to incorporate viral DNA into
the cell's chromosomes in step 4 above) has been developed and is currently in
clinical trials. The development of the other new inhibitors is still in its infancy.
Of course, researchers would ultimately like to find a cure and a vaccine for the virus,
rather than rely forever on treatments that only limit the extent of the infection.
Researchers are focusing on the attachment of HIV (in step 1) for developing
vaccines, and many researchers are hopeful that an effective vaccine will be found
within the next ten years.
RASMOL Files:
To view the molecules interactively, please use RASMOL. To download the pdb files
for viewing and rotating the molecules shown above, please click on the appropriate
name below. (Note: if you want to view multiple molecules simultaneously, please
download each file (.pdb) and save on your computer. Then open RASMOL outside
of Netscape.)


Reverse transcriptase (rt.pdb)
A short segment of an RNA template complexed with DNA- the substrate for
reverse transcriptase (rnadna.pdb)


Protease with protein substrate (hivprotease.pdb)
Protease with DMP323 inhibitor (protinhib.pdb)
Additional Links:






For more information about developments in the fight against HIV and AIDS,
see the "Why Files" on AIDS from the National Institute for Science
Education.
See the NOVA program that was broadcast on February 2, 1999. The program
looks at the AIDS research, and what scientists are learning about the immune
system.
This protease tutorial is interactive and offers an in-depth study of protease.
 Note: You need CHIME to view this tutorial. You can download
Chemscape Chime here. When you get to the home page for the
tutorial link, choose "Contents," then "Studying Protein Structures,"
then "HIV Protease."
Also be sure to see Kenyon University's HIV tutorial, which describes in detail
the inhibition of HIV at various points in the infection cycle.
This article describing how the HIV virus functions and reproduces itself was
provided by the Community Research Initiative on AIDS (CRIA).
The CDC National AIDS Clearinghouse offers daily updates on scientific and
political developments relevant to AIDS.
References:
Chen, M.S. et al. "Metabolism of 4'-azidothymidine," (1992) J. Biol. Chem., 267,
257-260.
"Good news about AIDS," National Institute for Science Education. The Why Files.
26 Mar. 1998. URL: http://whyfiles.news.wisc.edu/035aids/index.html.
Guex, N. and Peitsch, M.C. Electrophoresis, 1997, 18, 2714-2723. (SwissPDB
Viewer) URL: http://www.expasy.ch/spdbv/mainpage.htm.
"La zidovudina (AZT, ZDV, Retrovir)," National AIDS Treatment Information
Project. 1 June 1998. URL:
http://www.kff.org/archive/aids_hiv/natip/html/azt.html#A6.
Persistence of Vision Ray Tracer (POV-Ray). URL: http://www.povray.org.
Stryer, Lupert. Biochemistry. 4th ed., W.H. Freeman and Co., New York, 1995, 229230, 229-230, 835.
"Adults and children estimated to be living with HIV/AIDS as of end of 1997,"
UNAIDS 20 July 1999. URL:
http://www.us.unaids.org/highband/graphics/1997/report97/sld001.html
Vander, A. et al. Human Physiology, 7th ed. WCB McGraw-Hill, Boston, 1998, p.
700-727.
Weber, I.T. et al. "Molecular modeling of the HIV-1 protease and its substrate
binding site," (1989)Science, 243, 928. PDB coordinates as "HIV-1 protease complex
with substrate (theoretical model)," Brookhaven Protein Data Bank.
Yamazaki, T. et al. "Three-dimensional solution structure of the HIV-1 protease
complexed with DMP323, a novel cyclic urea-type inhibitor, determined by nuclear
magnetic resonance spectroscopy," (1996) To be published. PDB coordinates as
"HIV-1 protease-DMP323 complex in solution, NMR minimized average structure,"
Brookhaven Protein Data Bank.
Acknowledgements:
The authors thank Dewey Holten, Michelle Gilbertson, and Jody Proctor for many
helpful suggestions in the writing of this tutorial.
The development of this tutorial was supported by a grant from the Howard Hughes
Medical Institute, through the Undergraduate Biological Sciences Education program,
Grant HHMI# 71192-502004 to Washington University.
Copyright 1998, Washington University, All Rights Reserved.
Revised May 2001.