Download Chapter 9 – Catalytic Strategies (So we`ve talked about enzymes

Chapter 9 – Catalytic Strategies
(So we’ve talked about enzymes and that they act as catalyst to speed up
reactions, but we’ve haven’t really explained how all this process works. In this
chapter we’ll talk about the catalytic strategies for four different classes of
enzymes. )
(To me, this chapter is pretty cool because it shows what the heck is going on to
make a reaction that in some cases would take millions of years to happens to
occur in a matter of minutes or in some cases seconds.)
(Interestingly, although there are millions of different enzymes, they all have
similar strategies of converting a reactant to a product.)
Basic Catalytic Principles:
a. Enz binds a substrate
b. Binding controlled by correct “fit”
- Lock and Key or Induced Fit
c. Enz use 1 (or more) of the following strategies:
1. Covalent Catalysis - Reactive group in active site, forms temp. covalent
bond
2. Acid/Base Catalysis –Group in active site serves as an acid or base
- Promotes reactivity
(Thinking back to organic chemistry, there are several reactions that use and
acid or base as a catalyst to carry out the reaction. The same hold true with
enzymes, except that enzymes are even better. They can position the proton
or hydroxide in the perfect placement for the reaction to occur)
3. Proximity of Substrates – Brings 2 substrate close together in space
4. Metal Ion Catalysis – Can serve many functions
a. Create active Nu:
b. Serve as El+
(Probably the best way of seeing how these basic principles are used, is to look
at several enzymes and see how they catalysis applies.)
(Many enzymes perform same reaction, Enz specific to substrate: Many
enzymes work in a similar fashion and have structurally similar substrates.
However, since enzymes have to be really specific to their substrate, often there
are many enzymes that perform the same organic reaction but on a slightly
different substrate. As you can imagine, the active sites of these enzymes are
nearly identical, except for a few variations which make the enzyme specific for
its substrate. For this reason, our bodies contain thousands of nearly identical
enzymes, and many of them are very specific to their substrate as we saw in the
previous chapter.)
(Advantageous to study classes of enzymes: Therefore often we will study
classes of enzymes or groups of enzymes that have nearly identical active sites
but maybe more selective for one substrate then another enzyme in the same
class).
- Enz class = Enz perform same rxn, but specific to different substrates.
(It’s amazing that that you can have thousands of enzymes in the same class in a
cell but only one of these enzymes will react with a particular molecules … as
organic chemist, I know that we haven’t come close to nature is doing this)
(In this chapter we are going to cover classes of enzymes that perform
hydrolysis reactions, or reaction that add water to cleave a certain functional
group. The reaction we will consider will be breaking either a peptide bond or a
phosphate by through the addition of H2O).
(The enzymes we will cover will involve to overall schemes)
- Overview of Enz Hydrolysis:
(The first class of molecules we will study are the proteases.)
9.1 Proteases
(We’ve already seen some of these like Trypsin and Chymotrypsin).
*** ppt *** Proteases
(But by and large Proteases are enzymes that degrade proteins by cleaving
peptide bonds … like collagen in jello for example! There are a ton of these in
our bodies, but why are they so abundant?)
(Mainly for digestion but it also allows us to get rid of proteins that are no
longer needed or are damaged, and also in starving conditions are bodies will
actually Amino Acids as energy, but to do this proteins must be degraded first).
(So proteases are pretty important little enzymes)
i.
Role of proteases = degrade peptide bonds:
(The half-life of this reaction is usually 10 – 1000 yrs to perform at neutral pH,
but with a protease this drops to a matter of milliseconds)
- Proteases use both Covalent and Acid/Base Catalysis
ii. Catalytic Triad Proteases
- Use a 3 AA’s in active site (called triad) to carry out catalysis
Ex. Chymotrypsin
- Cleaves Carboxy side of Trp, Tyr, Phe, Leu, Met
(The pathway of Chymotrypsin is actually pretty cool. It actually is built as a
single peptide chain called chymotrypisinogen and forms a series of disulfide
bonds that hold the 3D structure together. This form is actually inactive and
doesn’t become active until another protease comes along and snips two
portion of the chain. It’s a clever way that our bodies have devised to make
sure that chymotrypisin is in the correct position and at a time of need when it
is activated)
- Key AA’s catalyzes 2 steps:
1. Ser 195 attacks carbonyl
(This is a covalent modification step. However, it leaves this Serine AA of
Chymotrypsin with an acyl group attached. We have to remove this for the
reaction to complete)
2. H2O attacks acyl on Ser 195
(So this is really the short end of it … but we said there is a catalytic triad: to
see how these other AA’s are used we must look at the organic reaction
mechanism).
(First, let’s take a look at that and see how Chymotrypsin uses these ideas of
both Covalent and Acid/Base Catalysis come into play)
- Mechanism for Chymotrypsin:
A. 2 AA’s make Ser 195 unusually basic:
- Asp 102, His 57 and Ser 195 = Catalytic Triad
- Function of Triad:
a. Asp 102 polarizes His 57 (the negative charge of Asp repells electron to
the other side of the histidine ring making it more negatively charge …
or a better base.)
b. His 57 acts as a base on Ser 195
c. Ser 195 alkoxide acts as a Nu on Substrate
(So these 3 AA’s really play together to make this active site work)
-
Arrow pushing:
O
R2
O H N
N H
1
:O
O H N
Ser 195
O
N
H
R1
N H
:O
Oxyanionic Hole
2
O
O
R2
Asp 102
His 57
O H N
:N
H
R1
O
N H
O
10, 11
3
Oxyanionic Hole
:O
R2
O:
O H N
N H
HO
O
O H N
R1
O
O
4
9
H O:
O
O H N
N H
R2 NH2
R1
O
O
O
O H
N
R1
O
O
8
H O
H
O H N
N:
O
R1
5, 6, 7
O
O
Steps:
1. Substrate binds
2. Alkoxide from Ser 195 attacks C=O of peptide
3. Tetrahedral intermediate stabilized by Oxyanion hole
4. His 57 transfers H+ to peptide amine (this makes it into a good leaving
group)
5. Peptide bond breaks
6. Amine portion released
7. H2O binds
8. His 57 removes H+ from H2O
9. OH- (previously H2O) attacks
10. Tetrahedral Intermediate forms
11. Carboxylic acid is released
*** ppt *** Label the Mechanism steps
N
N
H2
R1
O
(Now that is quite a process and interesting that this reaction happens much
less millisecond. As an organic chemist, I know that I can take a protein, boil it
in 1M KOH at 100 oC and still never get this reaction to work, but an
chymotrypsin can do this reaction in a fraction of a second. Why is this? It’s
really a very similar situation here?)
(The major difference between my boiling and this enzyme lies in the
Oxyanionic hole, which helps stabilize the tetrahedral intermediate.)
Oxyanionic Hole:
- See ppt
*** ppt *** Oxyanionic hole
- Stabilizes unfavorable tetrahedral intermediate
- Allows for protonation of peptide N = Good Leaving Group
(OK, so now we can explain how Chymotrypisin can easily cleave a peptide
bond, but one thing is missing how the heck does it get the selectivity it does?)
(Chymotrypsin typically cleaves Amino acids with aromatic side chains and some
long chain greasy hydrocarbons. That is why is specific for cleaving
Tryptophan, tyrosine, phenylalanine, leucine, and methionine.)
(It chooses these particular AA’s because just a little ways down from the active
site triad, and opposite of the oxyanionic hole, is a hydrophobic pocket called
the S1 pocket where the side chain of the AA being cleaved binds. So let’s take
a look at a diagram of this pocket.)
*** ppt *** Binding pocket of Chymotrypsin
a.
b.
c.
S1 Binding pocket:
Promotes selectivity of AA’s binding
Is rather large
Bulky AA’s fit best
(Of course Chymotrypisin is one example of many Proteases that exists, some
are more selective toward the amino acid to which they cleave and some are
less selective).
- For other Proteases (In the same class):
A. All contain similar catalytic triad AA’s
Ex. Trypsin and elastase
(These proteases actually contain 40% of the same AA’s as chrymotrypsin
and have nearly identical active sites … like the catalytic triad yet 60% of the
amino acids are different … why do you think that is?)
(This is for selectivity, as they will contain different hydrophobic S1 grooves
to better bind different AA’s)
B. S1 groove differ for selectivity
(Some S1 pockets will be smaller in size and or shaped differently so that it
chomps on different AA’s.)
(Very specific proteases will have as much as 6 pockets to pin down an exact
amino acid sequence … refer to ppt)
*** ppt *** binding pockets
(These proteases are often directed toward a certain damaging protein,
perhaps one of a virus or bacteria or something that will cause damage to
their precious cell. Instead, of introducing a protease that will destroy all of
the proteins with in cell, it creates one that will be pretty specific to the
pathogens evil protein).
(D-AA can’t be cleaved: Another very cool thing, is if you remember right
back in Chapter 1, all our AA’s in all protein are chiral, and for each Amino
Acid there a natural L enantiomer and an unnatural D enantiomer. Right?)
(Well one substitutes an unnatural D amino acid for an L one, the Protease
will have a hard time binding to it … but why?)
(In the case of an unnatural AA’s the side-chain of the AA’s point almost
exactly opposite of the binding pocket and probably into the side of the
protease cause a terrible interaction.)
(Consequently, scientists will actually incorporate unnatural D amino acids
into their proteins they make in labs to prevent them from get chomped on
by proteases… a pretty ingenious solution if you ask me).
(One other thing that I would like to point out is that you can have these
binding pockets before and after the point of cleaving … what do you think
this means?)
(Placing bind pocket before or after catalytic triad: Well if we said that these
binding pockets are what determines specificity, then it should true that if
the binding pocket or pockets are before or after the point of cleavage it will
determine if cleavage occurs at the carboxyl side or the amino side of the
amino acid. I bet this sounds a bit confusing but let’s see if we can clear it up
a bit).
*** ppt *** N selective Protease cleavage
*** ppt *** C=O selective Protease cleavage
C. Key point: Binding Pockets determine if Protease will cut on Carboxyl or
Amino side of an AA or AA sequence (or in middle of sequence)
*** ppt *** Rationalize Chymotrypsin
*** ppt *** Predict if the Protease will cleave Peptide at “C=O” or “N” end
iii. Other Classes of Proteases
(Most but not all proteases use this catalytic triad mechanism to cleave
peptide bonds. There are three other classes of proteases exist, which use a
different method of cleaving peptide bonds)
1. Cys - Works similar to Ser triad (work more like a diad, since Cys is more
reactive than Ser. This has to do with the Sulfur in the side chain of Cys
being a better Nucleophile than Oxygen in Ser. The result is that the Asp
used to activate the His base is no longer needed. So there is only a His
and Cys in this type of protease … it’s more like diad).
- Except:
a. Cys replaces Ser
b. No Asp
*** ppt *** Cysteine proteases
Ex. Papain – found in papaya (this is the protease we talked about earlier,
that chomps up jello)
Ex 2. Caspases – in mammals, many function including triggering apoptosis
2. Asp Proteases – Active site contain 2 Asp residues
- Asp’s polarize H2O and peptide bond
- Allows H2O to attack
*** ppt *** Asp Proteases
Ex. Retroviruses (ie HIV) often contain Asp proteases
(For example HIV contains HIV1-protease which is a protease needed for HIV
to work in cells. HIV1 protease is actually kind of a cool story. The way HIV
works is totally different from what we’ve learn so far. So, up to this point
you’ve heard DNA code for a protein, which makes RNA which then is
translated into a single protein. Well with HIV that is not the case. First of
all it doesn’t have DNA it only has RNA. Secondly, most of its proteins are
encoded on very long RNA strand. Therefore once transcribed HIV makes
many of its proteins in one long piece, with several proteins strung together.
HIV-1 protease has the job of cutting this long "polyprotein" into the proper
protein-sized pieces. )
3. Metalloproteases – Activates H2O to attack peptide bond
*** ppt *** Metalloproteases
Ex. Bacteria’sThermolysin contains Zn2+
- Involved in the degradation of flesh
iv. Protease Inh used as drugs
(As you can imagine, proteases shut down a very important property of cells,
that is the ability for cell to break down proteins whether it be a wide range of
proteins like in ones gut for digestion, or specific protein like in the case a
destroying a pathogen).
- Inh used to as medicine by stopping key proteases
(One of the most obvious that we just learn about would be the inhibition of
HIV1 protease, which all HIV or AIDs viruses need to survive. Currently, scientist
are working on great ways of shutting down HIV1 protease through inhibition
and have had some good successes. They unfortunately are encountering a few
difficult problems … that is that the HIV1 virus keep mutating and therefore the
binding site of HIV1 protease keeps changing. Therefore, the inhibitors become
ineffective over time. The second is that the inhibitors often “trick” a certain
protease into believing it is a protein, therefore these inhibitors often act of
other protease and as a result often cause side effect as a drug)
(Nevertheless, success has been made with HIV with a drug called Crixivan)
*** ppt *** Crixivan and HIV1 protease
9.2 Carbonic Anhydrases
(Remembering back to chapter 7, what again was the function of Carbonic
Anhydrase?)
- Converts CO2 + H2O -> H2CO3 (Carbonic Acid)
(Works very fast: As we mentioned before it one of the most rapid and most
efficient enzymes of the face of the earth).
(We said that the reaction hit diffusion limits, meaning that the reaction is only
slowed down by diffusion of both water and CO2 into the active site of the
enzyme).
(Anyhow, it has to be this fast because it catalyzes an equilibrium that is critical
for breathing, both in blood and in the inside of tissues).
(The absence of carbonic anhydrase in the blood would surely lead to death.
However, the absence other carbonic anhydrases, like in tissue cells for
example, lead to osteopetrosis (excessive formation of dense bone) and mental
retardation).
(Overall, carbonic anhydrase accelerates the rate of carbonic acid generation by
a million times, but how does it do this? Well you are about to find out…. Aren’t
you excited?)
i.
Zn2+ essential for activity of Carbonic Anh (what type of catalysis is
this?)
(In under 10 years after the discovery of Carbonic anhydrase, it was found to
have a Zinc atom in it. At the time this was a major breakthrough as no know
metals where thought to be a integral part of proteins. Now, it’s known that
about 1/3 of all enzymes contain some metal ion that aids in its activity. But at
the time this was a huge breakthough).
- Role of Zn2+ ion:
1. Acts as Lewis Acid
2. Lowers pKa of H2O
For plain H2O:
2H2O
H3O+ + OH-
pKA = 15.6
For H2O + Zn2+:
(This is crazy, by simply binding a zinc atom, the acidity of water increases by
100 million times. That is one huge difference, and it is only unique to
Carbonic Anhydrases. Simply, adding zinc to water won’t cause this effect as
it has something to do with it’s positioning in the enzyme.)
(If we consider Henderson-Hasselback eqn with this altered pKa of water, at
neutral pH in the blood or in cell, half of all water bound to the zinc ion, will
be deprotonated which is a surprising large percentage when you consider
that at pH 7 almost no water is deprotonated).
(So basically Zn ion allows an easy way to generate hydroxide, which is a
fantastic Nucleophile.)
(But what keeps this Zn ion in place,
- In Carbonic Anh, Zinc bound to 3 His residues
*** ppt *** 3 Histidines hold Zn atom in place
(These simply hold the metal in the perfect position for the next step in the
reaction).
- Conclusion: Zinc Ion helps generate hydroxide
ii.
Binding of CO2 positions perfectly for hydroxide attack.
(A hydroxyl isn’t going to do a whole lot of good unless it has CO2 to react with.
Another neat thing about Carbonic Anhydrase is that it has a binding site just
above the zinc atom.)
(It is positioned this way so that the hydroxyl atom formed from interacting
with zinc, which will lie slightly above zinc, is perfectly aligned to attack the
carbon of bound carbon dioxide).
- CO2 binds in hydrophobic pocket
- Aligned for hydroxyl attack
*** ppt *** Alignment of Carbonic Anhydrase Active Site
(So known all these key features of carbonic anhydrase, let’s see how it all
comes together to perform a reaction mechanism)
iii.
Mechanism:
Steps:
1.
2.
3.
4.
H2O bound to Zn is deprotonated
CO2 binds
Lone pair of hydroxide attack bound CO2
Water displaces bicarbonate
iv.
Carbonic Anhydrase Family
(A wide range of carbonic anhydrases exist outside of those in animals. In fact,
they are found in a range of different life and they are named according to their
structure which varies a great deal).
1.
2.
-
All known carbonic Anhydrases contain a Zn2+ atom
Types of Carbonic Anhydrases:
– Structurally similar to human carbonic anhydrases.
Found in animals, some bacteria, algae
- Zn bound by 1 His + 2 Cys (instead of 3 His as in )
Found in higher plants
- Allow plant to “trap” CO2 from the air
(We’ll learn about these types of enzymes latter next semester)
3. - Contains a left-handed helix
- Found in methane producing bacteria in hot springs
*** ppt *** Carbonic Anhydrase
9.3 Restriction Endonucleases
(As you may have learned in your biology classes, many viruses can actually
inject their DNA into a cell. Their DNA then gets confused with the host cells
DNA which leads to the production of viral proteins that essentially hijack the
cell and turn it into a factory for producing more viruses.)
(Host cells defense = restriction enzymes: As a defense strategy, cells have
grown smart and developed certain restriction enzymes, whose whole goal is to
seek out and destroy viral DNA by recognizing certain base pair then they
actually chomp up the DNA by hydrolysis similarly to proteases. And they do
this selectively to viral DNA).
(To do this restriction endonucleases much have a high degree of specificity on
two levels)
- Challenges:
1. Must detect recognition sequences
2. Must only degrade viral recognition sequences
i.
Reaction Performed: Hydrolysis of Phosphodiester bond
ii.
1.
2.
3.
Bond breaks between 3’ O and phosphate
Requirements:
H2O and DNA are required
Mg2+ or other divalent cation needed in active site
Ca2+ often found too
2 Asp residues: hold ion in place
(Often for the Restriction Endonucleases there are more than 3 metal ions)
iii.
Mechanism:
- Similar to Carbonic Anhydrase
- Mg2+ or Ca2+ drops pKa of H2O
- Arrow pushing (Don’t’ cover):
*** ppt *** Role of Mg2+/Ca2+ in the Active Site
iv.
Specificity
(So the mechanism follows roughly the same route as the Carbonic
Anyhydrases, but how the heck is this enzyme so specific to its target?)
(Well in part this is a result of viral DNA being somewhat unique in structure
through the process of evolution cells have taken advantage of this uniqueness.
So first we’ll talk about a common thread in many viruses DNA, and then we’ll
see how restriction enzymes have taken advantage of this).
A. Nature of Viral DNA: Inverted Repeats
Ex. EcoRV recognizes (a restriction enzyme of E.coli)
5’… GATATC… 3’
3’… CTATAG…5’
(It’s the same sequence flipped around … so is the major thing the enzyme
detects)
B. Binding of Inverted repeats to Active Site
- Involves key hydrogen bond contacts:
- For EcoRV:
5’… GATATC… 3’
3’… CTATAG…5’
- Where indicate H-bond w/EcoRV
- H bonding creates a “kink” in central AT’s
(This kink helps create a weak spot in the DNA backbone and helps
hydroxide attack off of the Mg2+ or Ca2+ ions).
- Kink helps cleaves DNA
- Overall picture: Refer to ppt
*** ppt *** Hydrogen Bond contacts
*** ppt *** Kink creates weak spots in DNA
- Specificity Occurs because:
1. Correct Inverted Repeat must be present
2. “Kink” must also occur
(So the real cool think about this is not only is the correct sequence needed
to bind to the Restriction Endonucleases like EcoRV and the enzyme uses this
sequence to kink the DNA and make it more reactive in the active site.
Without the correct sequence , binding would be difficult and kinking would
never occur and nearly impossible. Together, these combined features in
EcoRV make the enzyme over a million time selective for its target viral
RNA).
(Changes good host has same sequence: However, even though EcoRV and
other Restriction Endonucleases are insanely selective for the target DNA,
the chances are that the genome of the cell having that particular sequence
is good considering the vast number of nucleic acid bases all the DNA of the
organism. Therefore, host cell takes extra measures to protect its own DNA).
C. Protection of Native DNA
(Occurs by methylation of DNA these sequences that would be chomped up
by its own restriction enzyme.)
- Protected by methylation:
Ex. Methylation of Adenine in EcoRV:
(The result is that the hydrogen bond that could form before with adenine can
no longer form. This prevents kinking and ultimate stops the Restriction
Endonuclease from cleaving the DNA).
- Methylation prevents H-bonding and kinking = No cleaving
v.
Varations of Restriction Enz
- Almost all act by same mechanism (have an identical kinking mechanism
H-bonds changes to act on different sequences (which changes specificity)
(It’s likely that this enzyme is relatively recent in evolution history)
9.4 Myosins
- Enz that convert ATP + H20 -> ADP + P
- They drive motion in cells (aka carry out work)
(Every time you do work myosins make it happen: pick up you notebook you
have millions of this little enzymes chomping away at your ATP to give you
enough energy to make that movement. I don’t want to think about how
many of these things are going off carrying around some of the backpacks
that some of you do! You better thank your little myosins.)
*** ppt *** Intro to Myosins
(Myosins are typically quite elongated and at the ends have a Globular domain
were the active site is and where all the action takes place)
(So near the active site of each of these Myosin enzymes is dicationic
magnesium or manganese ion. What do you think the function of this ion is?)
- Near active site is either Mg2+ or Mn2+
(Actually you are wrong! It more has to do with binding and activating then it
does producing a hydroxide ion).
i.
Key Features of Myosin Active Site:
1. Role of Mg2+ or Mn2+
- Used for Binding
- Activates phosphate (act as a Lewis Acid)
(Another notable about this enzyme that right next to the far phosphate group
there is reservoir full of water in what is the active site).
(So if we are to modify the figure above, we’d can show the following)
2. Water pocket close to active site
3. Ser 236 near active site
- Serves as acid/base
ii.
Mechanism (show less detailed version):
1.
2.
3.
4.
5.
6.
Steps:
ATP binds
Ser 236 deprotonated by phosphate O
Ser 236 in turn deprotonates H2O
Hydroxide attacks
Pentavalent Phosphorus complex collapses
ADP and P leave active site
(So that’s the mechanism, but there is more too this. Somehow, this
mechanism or the process of going from ATP to ADP releases useable energy
by the enzyme. Otherwise if it didn’t then how in the heck could we use our
muscles or do any type of work whatsoever?)
*** Use Molecular Models to demonstrate below *****
(We can understand this from our mechanism. Take a look at the structure
labeled “D”. This structures 3D shape is actually considerably different from
the molecules “C” and “E” before or after. The reason is that in the structure
before the phosphate group is tetrahedral in shape, much like carbon is
normally. When an extra hydroxyl group adds it shifts to a triganal
bipyramidal arrangement. Ultimately, this leads to a huge geometric shift in
the whole myosin protein)
iii.
Mysosin conformational change
- Events:
1. Hydroxide adds to “C” to form “D”
2. “D” is triganal bipyramidal (considerably different in 3D shape to
tetrahedral “C”.
(It turn out this considerably different geometry actually shifts the oxygen of
the phosphate groups in close enough proximity to attract some nearby
Amino Acids around the active site by 2 angstroms)
3. Shift enables attraction of active site AA’s
4. Active site AA’s shift by 2 Å
(the movement of these active site AA’s causes a chain reaction which
completely moves a coil of 60 AA’s more than 25 Å … a huge distance!)
5. Chain reaction shift 60 AA coil 25 Å
*** ppt *** Conformational Shift
*** ppt *** Movie of conformational shift of Myosin