Download Lab 5: Proteins and the small molecules that love them

Life Sciences 1a Laboratory – Fall 2006
Lab 5: Proteins and the small molecules that love them
(AKA Computer Modeling with PyMol #2)
Goals: The objective of this lab is to provide you with an understanding of:
1. Catalysis
2. Small molecule inhibitors
3. HMG-CoA Reductase inhibitors
Introduction: As you have learned in the lectures, life is dependent upon
chemical reactions, such as the conversion of DNA monomers into DNA
polymers; RNA monomers into mRNAs, tRNAs and rRNAs; amino acids into
proteins; and the cleavage of HIV polyproteins into functional proteins. These
chemical reactions are thermodynamically favorable, however if these chemical
reactions were left to occur on their own, the rate would be too slow to support
life. Thus the rates of the chemical reactions need to be enhanced. In the
laboratory synthesis of Flexat to Diolat (Lab 5), you saw how chemical reactions
can be performed outside of the cell. Reaction rates can be accelerated through
the addition of heat, altering the pH of the solution, or adding catalysts – a
reagent(s) that speeds up a chemical reaction without being consumed. The
first two options are not very readily available inside a cell. The inside of a cell
cannot have it’s pH altered without having deleterious consequences for many
other processes and the temperature inside each cell is carefully maintained in
many organisms. However catalysts are available – usually in the form of
enzymes.
In this course, you have already been introduced to several enzymes that
catalyze chemical reactions – increase the rate of a chemical reaction without
being consumed themselves: DNA Polymerase, RNA polymerase, and the
Ribosome. Many other cellular chemical reactions are also dependent upon
enzymes. The breakdown of proteins, lipids, and carbohydrates ingested as food
are dependent upon enzymes. The post-translational processing of proteins
often require the assistance of enzymes. The biosynthesis of small molecules is
also dependent upon enzymes. These enzymes are able to catalyze chemical
reactions by employing several strategies: 1. Orientation and proximity – by
coordinating chemical reactants in their ideal orientations and in close proximity,
they can increase the rate of a chemical reaction by effectively increasing the
local concentration of the substrates and placing the atoms involved in the
chemical reaction in their ideal positions for the reaction; 2. Nucleophilicity and
Electrophilicity – through the precise positioning of amino acid sidechains, the
enzyme can alter the chemical features of individual atoms in a substrate to have
an enhanced affinity for positively or negatively charged reactants respectively.
Enzymes have another feature that distinguish them from chemical catalysts –
they are extremely specific in their choice of substrates. Enzymes bind
87
Life Sciences 1a Laboratory – Fall 2006
substrates in a region of the protein referred to as the ‘active site’. The active
site is where the chemical reaction will occur, but the shape and chemical
features (position of hydrogen bond donors and acceptors, hydrophobic, or
hydrophilic side chains) are such that only a very small subset of molecules will
be able to fit. This specificity ensures that the only very specific molecules will
be acted upon by any given enzyme. Because of the precise shape and features
of an enzymes active site, biochemical reactions can also be inhibited by
designing specific inhibitors that prevent the function of very specific enzymes.
In this lab we will investigate three different enzymes and see how the specific
positioning of enzyme sidechains allows for substrate specificity and increases
the rate of catalysis. Moreover we will see how two enzymes, HIV Protease and
HMG-CoA Reductase, can be beneficially inhibited by small molecule inihibtors.
Procedures: This lab consists of six sections; each section will utilize a PDB
coordinate file and an accompanying script. The questions will be due one week
from the day of the lab.
88
Life Sciences 1a Laboratory – Fall 2006
Section 1: Catalysis and Trypsin
•
Open up the folder titled “1_Catalysis,” then double-click on the PyMol file
labeled “Trypsin.pml.” This will automatically open up the PyMol program,
and load up the coordinates for the DNA structure.
In this section you will look at Trypsin to better understand how proteins orient
specific sidechains to bind specific molecules and catalyze chemical reactions. As
you learned in lecture from David, Trypsin is a key catalytic enzyme required for
breaking down proteins ingested as food. If trypsin were able to digest any
protein, it would be problematic as it would digest itself and once digested,
unable to function. Thus trypsin must accelerate the digestion of specific
proteins or peptides while sparing itself.
The first image you will see is of Trypsin in stick form. Notice the overall
‘globular’ nature of the protein. As a reminder, oxygen is colored red, carbon is
green, and nitrogen is blue.
•
Click off ‘Trypsin_Sticks’ and click on ‘Trypsin_Cartoon’.
Remember that a proteins structure often consists of multiple secondary
structure elements. In this image you can clearly see α-helices and β-sheets.
You will also notice many loops with less defined structures.
•
Click on ‘Substrate_Sticks’.
The substrate carbons are labeled purple to distinguish it better from Trypsin.
Notice how it interacts. As you will see later, the interaction of the substrate
with trypsin occurs at very specific positions, near specific residues.
1) The substrate is a folded protein, but trypsin only interacts with a portion of
it on the surface. One very prominent sidechain can be seen from the
substrate interacting with Trypsin. What is this amino acid?
•
Click off ‘Substrate_Sticks’, turn on ‘Substrate_Cartoon’.
Notice the region of the substrate that interacts with Trypsin. In this case it is a
loop region of the substrate – rather than an α-helical or β-sheet region.
•
Turn on ‘Cat_Triad’.
As you’ll recall from lecture, the active site of a protein is not always formed from
a continuous stretch of amino acids. The key amino acids can be quite distant in
89
Life Sciences 1a Laboratory – Fall 2006
the primary structure. In this image, three residues essential for Trypsins
function are highlighted.
2) Are these three key residues positioned next to one another in the primary
sequence of Trypsin?
3) Identify the three residues.
The three residues that are shown are essential for hydrolysis of the peptide
backbone of the substrate. Together these three amino acids are referred to as
‘a catalytic triad’. The precise positioning of these amino acids allows them to
efficiently hydrolyze the peptide backbone. It turns out that the residue closest
to the substrate is the one that performs the chemical reaction, however the
other two are essential for stabilizing the ionic charges that form during
hydrolysis.
•
Turn on ‘Cleavage_Site’.
The site of the substrate is now highlighted. The specific bond to be hydrolyzed
is located between these two residues.
4) What are the two amino acids shown on the substrate?
5) Which is the more N-term residue, more C-term residue? How do you know?
•
Turn on ‘Asp_189’.
Aspartate 189 is a key residue that determines substrate specificity. As you’ll
recall, aspartate is an amino acid with an acidic sidechain – thus at neutral pH it
is often deprotonated and has a negative charge.
•
Turn off all except ‘Asp_189’ and ‘Cleavage_Site’.
6) In this image what residues interact, and how do they interact?
•
Turn on ‘Binding_Pocket’.
Now we can see specifically how the substrate interacts with the threedimensional surface of the substrate binding pocket of Trypsin. Notice that Asp
189 is located at the end of a hydrophilic pocket (notice the blue and red).
Binding of the substrate in this hydrophilic pocket and Asp 189 are essential for
good substrate-enzyme interactions. This is the key portion that determines
substrate specificity for Trypsin.
90
Life Sciences 1a Laboratory – Fall 2006
7) Based on your knowledge of the substrate specificity pocket of Trypsin, what
other amino acid sidechains would interact well in the pocket?
Section 2: HIV Protease and its Substrates
•
Open up the folder titled “2_HIV_Substrate,” then double-click on the
PyMol file labeled “HIV_Protease_Substrate.pml.” This will automatically
open up the PyMol program, and load up the coordinates for the DNA
structure.
As you’ll recall from David’s lectures, HIV Protease is a key protein essential for
the post-translational processing of HIV polyproteins into several separate
proteins: matrix, capsid, nucleocapsid, protease, reverse transcriptase, and
integrase. Like Trypsin, HIV Protease specifically cleaves particular peptide
bonds in the polyprotein. This specificity is important in ensuring that the correct
proteins are formed. If cleavage was nonspecific, then many different proteins
would be produced with different N- and C-terminal amino acids, which could
alter their structures and functions significantly.
The first image you will see is of the two subunits that form HIV Protease in stick
form.
If you rotate the structure, you’ll notice a ‘hole’ between the two subunits. This
hole is the active site of HIV Protease and is formed by the interaction of the two
Protease subunits with one another.
•
Turn off ‘Sticks_A’ and ‘Sticks_B’, and turn on ‘Cartoon_A’ and
‘Cartoon_B’.
The active site is less obvious in the cartoon form, however it is still present. In
this image, the structures of the two individual subunits is much more apparent.
HIV Protease is formed from two identical HIV protease subunits. Thus if we
were to look at the primary sequence of the blue subunit, it would be identical to
that of the green subunit. More importantly, if these two subunits did not
interact with one another, HIV Protease function would be severely impaired as
we will see later.
8) In what orientations do the two subunits interact with one another?
•
Click on ‘Substrate’
The substrate sits in a pocket formed by the junction of the two HIV Protease
subunits. Interactions between sidechains of both subunits and the substrate
91
Life Sciences 1a Laboratory – Fall 2006
are key for defining substrate specificity. Neither subunit alone is sufficient to
specifically bind solely the proper substrate.
•
Click on ‘Asp_25’.
Remember that catalysts are not consumed in the chemical reaction they
catalyze. In the case of HIV Protease, this is abundantly clear as Protease helps
coordinate a water molecule to cleave the polyprotein backbone. Two of the key
residues for this are Asp 25 and Asp 25’. The prime is used to notate that the
residues are on separate, but identical subunits. Asp 25 and 25’ are able to
enhance the hydrolysis of the HIV polyprotein by first making water a better
nucleophile (positive charge loving) through the deprotonation of water, while
simultaneously making a specific carbon atom in the polyprotein a better
electrophile (electron loving) by protonating a carbonyl oxygen. For more detail
see David’s lecture notes.
9) Draw the base catalysis and acid catalysis reaction performed by HIV
Protease (hint, see David’s lectures).
Now if you hadn’t noticed, you’ll see that two Asn residues are shown rather than
Asp residues. This was a key change made for the purposes of crystallizing the
enzyme-substrate complex. If the normal protein had been used, it would have
cleaved the substrate. This mutant form of HIV Protease is still able to bind the
substrate, however it is unable to catalyze the hydrolysis reaction. The enzymesubstrate complex can thus be crystallized and the structure could be solved.
10) Why is the mutant form of HIV Protease shown unable to hydrolyze the
substrate?
•
Turn on ‘Ile_50’ and ‘Water’.
The the attacking water molecule is positioned by coordinating it in a precise
position with the backbone nitrogens of two Ile residues. This places the water
in an ideal position to be acted upon by Asp25, and for the water to
simultaneously attack the substrate backbone.
•
Click off ‘Water’, ‘Cartoon_A’, and ‘Cartoon_B’. Turn on ‘Pocket_Residues’.
As mentioned earlier, the binding pocket is key for ensuring that the enzyme acts
upon the proper substrates. In this model the sidechains of HIV Protease that
help form the substrate-binding pocket are shown. Notice that the binding
pocket is made up of residues from both subunits.
92
Life Sciences 1a Laboratory – Fall 2006
11) Do you notice anything particularly interesting about the order of the amino
acids lining the binding pocket from both subunits (hint, see question 8)?
•
Turn on ‘Binding_Pocket’.
Now when we look at the surface of the binding pocket, we can see the
interaction of the substrate more clearly. Notice the nice fit of the substrate, the
interactions that form as well as the multiple openings in the pocket. This is a
key feature as HIV Protease acts upon several different protein sequences and is
thus able to cleave several different HIV polyproteins with different sequences
into the final protein products.
12) Based on the surface representation of the binding pocket, is HIV Protease
as selective or less selective of its substrate in comparison with Trypsin?
In the next two sections we will examine two different protease inhibitors.
93
Life Sciences 1a Laboratory – Fall 2006
Section 3: HIV Protease Inhibitors – Indinavir
•
Open up the folder titled “3_HIV_Indinavir,” then double-click on the
PyMol file labeled “HIV_Protease_Indinavir.pml.” This will automatically
open up the PyMol program, and load up the coordinates for Hemoglobin.
If HIV Polyproteins cannot be cleaved into individual proteins, the HIV life cycle
will be disrupted. Thus a key class of drugs used to inhibit HIV replication are
protease inhibitors. These molecules have been specially designed to bind in the
active site of HIV Protease and thus prevent Protease from acting upon it’s
normal substrate.
The first model you will see is of HIV Protease in cartoon form.
•
Turn on ‘Indinavir’.
The Protease inhibitor Indinavir is now shown in stick form. Recall that it is
positioned in the active site formed by the interaction of two protease subunits.
Indinavir is referred to as a ‘competitive inhibitor’ meaning it competes with the
normal substrate for binding.
•
Turn on ‘Asp_25’, ‘Ile_50’, and ‘Water’
Notice that Indinavir is coordinated in the same position as the normal HIV
Protease substrate and interacts with the key enzymatic residues, Asp25 and
Ile50, in the same way. Also notice that in this model ‘Asp_25’ is really
represented by Asp residues rather than Asn residues. If you zoom in on
Indinavir, you will notice that the backbone chain is not the same as a normal
peptide backbone. This change prevents it from being cleaved by the protease.
13) Why could a crystal structure be made of Indinavir with normal HIV
Protease?
•
Turn on ‘Binding_Pocket’.
When we look at the binding pocket we see that Indinavir fits well and makes
many positive interactions with the HIV Protease binding pocket sidechains.
Thus it is clear that it is able to form many of the same positive interactions as
the normal substrate, but is uncleavable.
14) For Indinavir to be an effective inhibitor, why is it important that it cannot
be cleaved?
94
Life Sciences 1a Laboratory – Fall 2006
Section 4: HIV Protease Inhibitors – Dupont
•
Open up the folder titled “4_HIV_DuPont,” then double-click on the PyMol
file labeled “HIV_Protease_DuPont.pml.” This will automatically open up
the PyMol program, and load up the coordinates for Top7.
As you saw in the previous section, Protease Inhibitors can be designed that bind
in similar fashions as natural substrates. However in this section we will look at
a newer Protease Inhibitor with enhanced binding affinity and study why it binds
better.
•
Turn on ‘Dupont’.
It is clear that the DuPont inhibitor binds in the active site of HIV Protease just
as Indinavir does. However if you look at the structure more closely, you’ll
notice a key difference in the molecule. At the center of the molecule is a 7member ring. Ring structures are inherently more rigid than linear structures.
15) What thermodynamic effect will the presence of a ring in the inhibtor have
upon its binding to HIV Protease?
•
Turn off ‘Cartoon_A’ and ‘Cartoon_B’. Turn on ‘Asp_25’ and
‘Pocket_Residues’.
In this model we still see that the DuPont inhibitor is well coordinated with the
pocket residues and the key Asp25 catalytic residues. No clear explanation can
yet be seen for the increased binding affinity of this molecule over Indinavir.
•
Turn off ‘Asp_25’ and ‘Pocket_Residues’. Turn on ‘Binding_Pocket’.
The same nice fit is seen when the binding pocket surface representation is
shown.
•
Turn off ‘Binding_Pocket’, turn on ‘Ile_50’, ‘Asp_25’ and ‘Water’.
The carbonyl oxygen connected to the 7-member ring in the middle of the
molecule is coordinated with Ile50 and Ile50’. Normally Ile50 and Ile50’
coordinate a water molecule to attack the substrate. By specifically placing a
carbonyl oxygen on the inhibitor to coordinate with Ile50 and Ile50’, one extra
water molecule is liberated from interacting with the protease.
16) What will be the thermodynamic effect of liberating the water molecule
normally used to attack the substrate upon HIV Protease binding?
95
Life Sciences 1a Laboratory – Fall 2006
Section 5: HMG-CoA Reductase and HMG-CoA
•
Open up the folder titled “5_HMG_Substrate,” then double-click on the
PyMol file labeled “HMG_Substrate.pml.” This will automatically open up
the PyMol program, and load up the coordinates for bulk water.
For more information about HMG-CoA Reductase, see the appendix attached to
Lab 5 and more information is included with Lab 7. Cholesterol is synthesized in
the cell through a series of chemical reactions, each using a different enzyme.
One of the key steps in cholesterol biosynthesis is the conversion of HMG-CoA to
Mevalonate using HMG-CoA Reductase. This is considered the first committed
step as once a molecule of HMG-CoA is converted to mevalonate, it is committed
to the cholesterol biosynthetic pathway.
17) Uncatalyzed, the reaction converting HMG-CoA to Mevalonate is extremely
slow. HMG-CoA Reductase accelerates this reaction markedly. On a reaction
energy diagram, draw the graph that would be expected for the spontaneous
conversion of HMG-CoA to Mevalonate in the presence and absence of HMGCoA Reductase. Include in your drawing ΔG‡ and ΔGrxn
The first model you will see is of a complete HMG-CoA Reductase molecule.
18) How many individual polypeptide chains form a single HMG-CoA Reductase
protein?
•
Turn off ‘Full_Tetramer’ and turn on ‘Interface_A’ and ‘Interface_B’.
While the full structure is interesting, it is a bit cumbersome to use. In this
model you can see the interlinking fit between two of the HMG-CoA Reductase
subunits.
•
Turn off ‘Interface_A’ and ‘Interface_B’. Turn on ‘Cartoon_A’, ‘Cartoon_B’,
‘Coenzyme_A’, and ‘HMG’.
This model shows the substrate interacting with the enzyme. Normally HMG and
CoA are connected as a single molecule when binding to the enzyme. However
in this case they are shown already cleaved and separated.
•
Turn on ‘NADP’.
For each molecule of HMG-CoA, two molecules of NADPH are used to reduce
HMG-CoA to Mevalonate and CoenzymeA. In the process, these two molecules
of NADPH are oxidized into NADP+.
96
Life Sciences 1a Laboratory – Fall 2006
19) In one of Dan’s lectures, he spoke of a specific oxidation and reduction
reaction with regards to protein structure. What other example of oxidation
and reduction have we seen in the course? (hint, think perms).
•
Turn off ‘Cartoon_A’ and ‘Cartoon_B’. Turn on ‘Binding_Pocket’.
Notice how the HMG interacts well with the binding pocket, but the CoA doesn’t
interact much. Thus much of the binding of HMG-CoA to HMG-CoA Reductase is
driven by the HMG portion, and not the CoA portion of the molecule.
97
Life Sciences 1a Laboratory – Fall 2006
Section 6: HMG-CoA Reductase and Fluvastatin
•
Open up the folder titled “6_HMG_Drug,” then double-click on the PyMol
file labeled “HMG_Drug.pml.” This will automatically open up the PyMol
program, and load up the coordinates for bulk water.
As you’ve already learned, Fluvastatin inhibits the function of HMG-CoA
Reductase, and thus decreases the biosynthesis of cholesterol.
In this model, only two of the four subunits of HMG-CoA Reductase are shown.
•
Turn on ‘Fluvastatin’
You’ll notice that Fluvastatin has a moiety that is very similar to HMG. This
portion is key for its ability to bind in the active site of HMG-CoA Reductase. Also
observe that similar to HIV Protease Inhibitors, Fluvastatin cannot be reduced to
form two separate molecules. Thus an individual molecule of Fluvastatin can
inhibit HMG-CoA Reductase for a long period of time.
•
Turn on ‘Binding_Pocket’.
Observe the binding of Fluvastatin. As mentioned previously, the HMG moiety of
Fluvastatin allows it to bind in the active site of HMG-CoA Reductase. However
for the molecule to be useful as an inhibitor, it needs to bind with greater
affinity.
20) What effect will the relative affinity of an enzyme for an inhibitor versus its
natural substrate have upon the concentration or dose of the inhibitor
required to inhibit normal enzyme function?
As you’ll recall from the appendix of Lab 5, all Statins also possess hydrophobicring structures. These ring structures interact with a hydrophobic patch near the
active site of HMG-CoA Reductase, thus increasing binding affinity. The normal
substrate, HMG-CoA lacks the hydrophobic patch and thus does not form this
interaction.
21) Thermodynamically, how does the presence of the hydrophobic rings
increase binding affinity? Is entropy or enthalpy more affected?
98