Download Control of Enzymes

Chapter 10 – Regulatory Strategies
(Last chapter I mentioned that there are these whole classes of enzymes that
basically have the same active site, they catalyze the same exact reactions, and
they and many of the same enzymes are actually present in the same exact cell.)
(Granted that we mentioned many of these enzyme are extremely specific to
their substrate, but still for body to be working properly, these enzymes need to
be heavily controlled. Most of the enzymes have a time and place for their
activity. For example, Myosins can’t be chomping up valuable ATP if you’re are
sleeping … that’s just inefficient … now if you are being chased by a bear then
perhaps it’s time for your Myosin to kick into action. But until then its wasteful
to use up your bodies precious energy on nothing.)
(So how does your body control all these enzymes so that they available only
when needed. Or in scientific terms how do our bodies “regulate” enzymes?)
(That’s what this chapter will be about. We’re going talk more about the on
and off switches to enzymes).
- Content: Control of Enzymes
(In a nutshell there are 5 different strategies that living organisms use to control
enzymes.)
i.
5 strategies to control Enz:
1. Allosteric Control
- Allosteric Site:
a. Binding site that controls activity of Enz
b. Not active site
- Allosteric Effector binds to site to turn on or off Enz:
(So if you think about it, allosteric site are kind of nice because they more or
less have an on/off switch. In many cases, one of the products of the
reaction actually will serve as an allosteric inhibitor. Why do you think that
is often the case?)
(This is because if the product actually accumulates to high levels, it is not
likely being used very much by the body any more. For this reason it much of
it hangs around the enzyme and can then shut off the enzyme as it isn’t
needed anymore).
(The second strategy we’ve talked quite a bit about already. And that is this
idea that the instead of one single enzyme that catalyzes one reaction for
many different substrates, we actually have many enzymes of the same class
that perform the same reaction on many different substrates. Or to say it
simply enzymes have substrate specificity).
2.
-
Isozymes
Different Enz catalyze identical rxn
Done at different rates
Controls rxn rate in different cells
3. Reversible Covalent Modification
(So some enzymes can be activated by forming a covalent bond with a small
group. This is idea is similar to allosteric control but results in a new covalent
whereby, allosteric interaction never form a covalent bond).
(Anyhow, modifying the enzyme then either turns on or turn off its activity.
However, this covalent bond can only be temporary or else the enzyme will
be permanently fixed into an on or off state).
(Often a phosphate group from something like ATP, will be attached to the
enzyme to activate or deactivate the enzyme. So here is an instance where
ATP is not actually used for energy but it instead used to either turn an
enzyme either on or off).
- Group covalently added to activate/deactivate Enz:
(The instance above is a very common one. There are many types of
molecules that perform the function of Enzyme B and take ATP to add it to an
Enzyme. These particular Enzymes are referred to as kinases. Enz C, which
cleaves off the phosphate group is referred to as a phosphatase).
4. Proteolytic Cleavage
- Enz activated by cleaving portion of peptide
(We saw this with chymotrypsin, where the enzymes peptide chain had to be
chomped into 3 portion before it was made active. So far we’ve talked about
enzymes being able reversibly go back and forth between active and inactive
forms. With these the change is permanent).
- Once cleavage activation is permanent
(Well that is until the enzyme is damaged somehow and is then recognized
by the body as trash and is degraded).
(Many of the digestive enzymes that degrade protein, such as the INN and
Out hamburger you ate for lunch are activated in your gut like this. When
you cut yourself your body forms blood clots. The ways that this works is
that you have quite water soluble clotting proteins in your blood. The
moment a clot in needed, these proteins are cut open to expose their
hydrophobic interior. Naturally, the want to glom onto each other and that
results in clotting. As more blood passes through this area (such as a cut)
more are activated and the glomming continues until a patch of blood clots
are formed to seal the wound.)
5. Controlling Amounts of Enz
(The last way or regulation we’ll talk about is this controlling of the amount of
Enz present to do the job. Less enzyme simply means that the reaction can’t
happen as fast. )
- Less Enz = less rxn happening
10.1 Allosteric Control
- Example: Aspartate Transcarbamoylase (ATCase)
- Makes CTP (which is like ATP)
*** ppt *** CTP role in cells
i. Reaction:
ii. Allosteric Control
-
ATCase inhibited by CTP
- Key: where
stands for Inh
(Why do you think it would do this?)
(This is how it controls whether or not CTP needs to be produced. If CTP is
being used in a cell then it will rapidly be depleted from the cell and not in
high enough concentration to inhibit the Aspartate Transcarbamoylase,
however, if it stops being used then more CTP will build up in the cell. As
time continues, it will eventually build a level to where it is very abundant.
At which it will bind to Aspartate Transcarbamoylase to stop its own
production.)
(It will stay this way until CTP becomes scarce again, and won’t be at high
enough concentration to inhibit Aspartate Transcarbamoylase and then CTP
production will start again!)
- 2 scenarios:
1. If CTP abundant: Inhibits Asp Tran = Signal for no more CTP made
2. If CTP scarce: Asp Trans starts production = Signal to make CTP
(OK, so hopefully that makes some sense, but the bigger question is and one
that probably takes more explanation is how does this enzyme do this?
We’re now going to look at this in detail).
iii. Subunits
ATCase has 2 types of subunits:
1. 6x catalytic subunit or “c chains” (or the subunit that actually catalyzes
the reaction and contains the active site.)
- Catalyze rxn
(So let’s take a closer look into the reaction that is going on and what the
catalytic subunits look like)
*** ppt *** C Chains of ATCase
2. 6x regulatory subunits “r chains” (contains allosteric site and control the
enzymes whether the enzyme is on or off)
-Contain Zn atoms
- Turn on/off Enz
(I could attempt to draw this but it probably won’t be pretty . So we’ll put a
note to refer to powerpoint)
- For structure: refer to ppt
*** ppt *** Structure of ATCase
iv. T and R states of ATCase
(Remember back to the hemoglobin chapter, what did the T and R state of
Hemoglobin mean?)
(So, in the T or “tensed” state Hemoglobin is not the flexible and the binding
of Oxygen was difficult … but the in R or “relaxed” state binding of oxygen
was fantastic.)
(Well this same idea applies to ATCase too. Like hemoglobin ATCase has
both a T and R state. )
- T or “tensed” state: Asp and Carbamoyl phosphate substrate do not bind
well.
- R or “relax” state: Asp and Carbamoyl phosphate binds well
- Key point: Binding of a set of substrates allows each additional substrate
to bind better (or more promotes the R state)
- Or ATCase participates in cooperative binding
(Remember that there are a total of 6 catalytic units. That means a total of 6
pairs of substrates can bind – which would really promote the R state).
(This would look like the following)
*** ppt *** Aspartate and Carbamoyl Phosphate Binding Promotes R State
(Basically what is happening is that when the catalytic subunit go into an R
state there is a space that is created where the active site can be better
accessed by the substrates. This allows much easier binding).
(So this is really cool because it means that also if there is an excess of Asp
around it promotes CTP synthesis. And if there is a lot of CTP around then it
shuts off CTP synthesis, so depending on the situation the ATCase can adapt).
***ppt *** Favoring T state or R state
(So really the next question is now, how does CTP shut down the activity of
ATCase?)
v. CTP and T and R states ATCase
About R chains:
(Each of the regulatory subunits or r chains are joined on one side to a
portion of the catalytic subunit an on the other side to another r chain. So
essentially two r chains meet. At the point where they meet it serves at a
hinge that enables the ATCase to smoothly transfer from the T-state to the Rstate upon binding of the substrates).
- 1 side attached to C chain
- 1 side attached to R chain
- R chains serve as a hinge
Key point: CTP binding prevents r chain hinge activity promoting T state
(So in essence, if you imagine almost all doors have hinges. Well CTP would
serve as a door jam that prevents the function of the hinge. So CTP binding
essentially is jamming up this hinge so it can’t move into the R-state. As a
result of this, substrate has a hard time binding to the concealed active site).
vi. Effect of ATP on ATCase
(Oddly, ATP which somewhat close in structure to CTP can also bind to the
allosteric site as well. However, when it binds it has virtually no effect on the
ATCase. Really the only thing is does it block the allosteric site so that CTP
can’t bind and act as a hinge jam.)
- ATP binds to allosteric site
- Blocks site so CTP can’t bind (an inhibit the enzyme)
(So in effect, ATP promotes ATCase activity ultimately leading to more CTP.
Now why the heck after many years of evolution would the enzyme behave
this way?)
(It turns out that there are 2 reasons)
- Reasons for ATP binding:
1. CTP to ATP ratio is off in cells
(The ratio of CTP to ATP needs to be at a certain level in cells for the cells to
function properly. If ATP is much more abundant then CTP, then for
whatever reason the ratio is off, even though there is enough CTP to
efficiently shut off ATCase).
2. ATP associated w/growth
(If the cell is growing and is about to divide a lot of work needs to be done
and CTP will also be needed in abundance to make phospholipids and such
for the rapidly growing cell. For this reason, the cell will produce a lot of ATP
for energy and it will still need to produce a bunch of CTP too. So you don’t
want the CTP production being shut off so ATP in a growing cell will prevent
that from happening).
10.2 Isozymes:
(As mentioned briefly before this is enzymes that perform an identical
reaction but are composed of completely different amino acid sequence and
they are encoded on different portions of DNA)
(So why would living organisms have different forms of the same enzyme,
that seems like it would be a bit redundant?)
(Well not all tissues have the same demands. Like take your muscles for
example, muscle cells are meant to lift heavy objects and perform activities
such as running and require an enzymes that will enable these cells to utilize
ATP very efficiently. Cells in the retina of your eye will never worry about
things like running or lifting heavy weights, but need to use ATP for work in
other but less demanding ways.)
(So for this reason, the body has built two different form of an enzyme with
the same function. One is meant for the muscle and one for the retina cells.)
(Let’s take a look at a specific example)
Ex. Lactate Dehydrogenase (LDH)
- Energy production: Glucose -> ATP
- 2 isozymes:
1. H – good for aerobic conditions (in cells that obtain a lot of O2)
(These are like Heart cells – as it is close to the lungs -, Kidney, Red Blood
Cells)
2. M – good for anaerobic conditions (in cells that are often depleted of O2)
(In muscle cells and Liver cell which both do some really hard work and
often need aerobic conditions to produce energy)
- LDH exist as a tetramer
(This means that 4 individual isozymes will come together to form the
tetrameric Lactate Dehydrogenase complex)
- Possible combinations: H4, H3M, H2M2, HM3, M4
(Depending on the cells demand for aerobic versus anaerobic respiration that
it experiences will determine which combination or combinations of H/M
isozyme that it will have)
*** ppt *** H/M tetramers for Various Cell Types *** 2 clicks ****
10.3 Covalent Modification
(We’ve already discussed and will discuss more that phosphorylation is major
method of protein modification. But let’s briefly look at several other methods
of protein modification)
*** ppt *** Common Types of Protein Modification
(For this chapter we’ll focus mostly on phosphorylation because it is by far the
most frequent process that you’ll encounter in cells).
i.
Phosphorylation in cells
- Kinases: Use NTP’s in cell to add P(circle) to Enz
Note: NTP can be ATP, CTP, GTP or TTP
- Phosphatase: Cleave P(circle) from Enz
(This process of phosphorylating and dephosphorylating proteins is
extremely common in cells. In fact, nearly 30% of all eukaryotic proteins
under phosphorylation/dephosphorylation which is an insane percentage).
(But why is phosphate so heavily used in cells? What makes it so valuable to
cells)
(Virtually every metabolic process or process that controls metabolism, or
the processes of life is controlled by phosphorylation. So this is a big deal).
(Oddly, almost all kinase and phophatases fit into 1 of 2 classes. So, really
it’s pretty much only one real type of kinase that is running all of our
metabolisms. Granted within this class of enzymes, there is some diversity,
but the active site of all the enzymes within this class is nearly identical).
ii.
Classes of Kinase/Phosphates:
1. Ser/Thr Kinase/Phosphatase: Add/Remove P(circle) from OH groups on
certain Ser/Thr side chains.
- Most common
2. Tyr Kinase/Phosphatase: Add/Remove P(Circle) from OH groups of Tyr
- Less common
- Mutations lead to cancer
iii.
Specificity of Kinases
(Kinases can either be very specific to their target or not very specific at all,
depending on the need in the cell. For this reason kinases are classified as
being ):
a. Dedicated Kinases – specific to a single protein or small set of proteins
b. Mulfi-functional Kinases - modify many targets
(Whether a kinase will be dedicated or multi-functional depends on the
consensus sequence of amino acids that it recognizes. The longer the
consensus sequence, the more specific the kinase).
- Specificity depends length of consensus sequence
Ex. Arg-Arg-Ala-Ser more specific than Ala-Ser
4x *** ppt *** PP2A and PP1 + My pH.D. thesis
iv.
Useful Properties of NTPs (ATP, CTP, ect) and P(circle):
1. G of Phosphorylation = -12kJ/mol
(This is fairly extremely exergonic reaction. Meaning that the reaction will
takes place quite easily in one direction especially with the aid of an enzyme)
2. 2 Neg charges
(This can have a phenomenal effect on an enzymes 3D shape. These two
negative charges will repel other negatively charged side chains such as
aspartic or glutamic acid groups in the enzyme. Likewise, it will attract
positively charged groups such as Lysine or Arginine. The change cause a
domino effect that changes the whole 3D shape of the enzyme to either
promote or deter an active form)
- Repels Asp or Glu groups
- Attracts Arg or Lys
- Helps change overall shape of Enz
3. Can form >3 H-bonds
- Creates new interactions = change in 3D shape
4. Phosphroylation/Dephosphorylation rapid or slow
(So depending on the enzyme being phosphorylated or dephosphylated the
whole process can take less that a fraction of a seconds or hours depending
on the protein at hand. This gives the cell real control over how rapidly or
slowly it needs for a process to occur).
- Gives cell control over speed for activating/deactivating certain Enz’s.
5. NTP’s in high conc. when cell is energized
(Since there is a lot of ATP or CTP around when the cell has been producing
and storing energy for a while, then the activation of enzymes can be tied to
the cells state. So suppose a cell has been sitting around for a bit and really
been spending most of it is time making ATP. There should be a lot of ATP
around in the cell. Since there is a lot of ATP available the proteins can easily
be phosphorylated by kinases. So a highly phosphorylated state of proteins
can be related to the cell as kind of a resting state. )
- When NTP’s abundant more Enz in phosphorylated state
(Imagine a different situation where ATP is needed to make a muscle work,
like when lifting weight or running. In this situation ATP is being burned up
at an astonishing rate. During this period of time, the concentration of ATP is
going to be much lower simply due to the lack of ATP available to
phosphorylate enzymes. As a result, many of the enzymes will be
dephosphorylated by phosphatase but will not be phosphorylated so the
relative ratio of dephosphorylated to phosphorylated enzymes will be much
greater).
- When NTP’s less abundant more Enz in dephosphorylated state
v.
Phosphorylation Cascades
- Results in amplification of signals
- Ex. Hormone binding to cell
2 x *** ppt *** Amplification in Signal
*** ppt *** Plastics as Estrogen Mimics
10.4 Proteolytic Cleavage Activation
- Central Idea: Enz inactive until cleave (it is has to be cleaved at the
correct positions)
- Naming:
a. Uncut Enz = zymogen or proenzyme
b. Active Enz
i.
Common Examples:
1. Digestive Enz
(There are a whole slew of these enzymes that are inactive until they hit your
stomach then they are turned on. Let’s see an example of a few and how
they interact via cleavage).
*** ppt ** Examples of Digestive Enzyme Activated by Peptide Cleavage
*** ppt *** Steps involve in Activation
- HCl usually activation
- Active digestive Enz activate other zymogens
2. Blood clotting – Cascade of cleavages
3. Insulin – Removal of a portion on peptide
4. Collagen – protein in skin and hair
5. Programmed Cell death – Activated by cleavage and result in kill cell
ii.
Digestive Enz
- Ex 1. Chymotrypsin
(Last chapter, we talked about the activity of chymotrypsin in great detail as
to how it works and now we’ll talk about exactly how cleaving the peptide
into 3 portions activates it).
- Zymogen = chymotrypsinogen = 1 continuous strand of AA’s.
- Active Enz = 2 to 3 strands AA’s
(If you can visualize this, if we are going from one strand to 2 to 3 strands
there had to be 1 to 2 cuts somewhere)
*** ppt *** Cuts to activate Chymotrypsin
Activation of Chymotrypisin:
- Triggered by cleaving AA’s 15-16
(How the heck could cleaving one 15 AA fragment allow a complete activation
of the whole enzyme? Let’s take a look at the details behind what is happening
here).
1.
2.
3.
4.
Details:
Ile16 (new N terminus) form new ionic bond w/Asp 194
Many AA’s to move from interior to exterior of Enz
Movement creates binding pocket and oxyanion hole
Other subtle changes
(So to summarize this cleavage is essentially opening up the binding pocket
needed to bind the amino acid side chains of the substrate peptide. In
addition, the oxyanion hole forms too, remember what that does? It
stabilizes the tetrahedral intermediate).
(Without a binding pocket or oxyanion hole, the protease activity of
chymotrypsin is diminished completely to nothing!)
(Other proteases have similar activation mechanisms.
iii.
Blood Clotting
- Involves a Enz cascade rxn
A. About Enz cascades:
1. Achieve rapid response
2. Similar to effect of signal amplification (which we saw with
phosphorylation)
Ie 10 enz activate 10 more which activate 10 more
=10,000 amplified
3. Usually few Enz trigger cascade
B. Blood Clotting is initiated by 1 of 2 ways:
1. Intrinsic pathway – Trauma to blood vessels exposes endothelial cells.
The cells anionic surface initiate Enz cascade
- Less common (but more direct pathway)
2. Extrinsic Pathway – Trauma results in production of tissue factor (TF)
which initiates cascade
(usually it is the endothelial blood cells that actually do the producing of the
tissue factor as a response to the cells being damaged).
- Both pathway cascades converge (which gives the same outcome … the
production of platelets).
*** ppt *** Intrinsic and Extrinsic Cascade Pathways
(We know a lot of what happens at the convergence point of these 2
pathways. This is the process where the blood clots are produced or more
specifically the process by which Fibrogen is converted into a crossed linked
fibrin clot or a blood clot)
C. Fibrigen to Fibrin Blood Clot
(Before we see how all this works let’s take a good look at Fibrigen first, in
order to get an idea of how this all is going play out).
*** ppt *** Structure of Fibrinogen
- Conversion catalyzed by Thrombin and Factor XIII
1. Role of thrombin – Cleaves 18 AA piece of subunit A and 20 AA’s of
subunit B.
- Portion removed called fibrinopeptides
- Remaining protein = Fibrin
- Effects:
a. Remaining tips of A-subunit bind to a groove of -subunit
b. This results in polymerization of Fibrin
(Note that the fibrinopeptides whole purpose it to block A-subunit from
interacting with the -subunit. Once they are removed then these 2 subunits
can freely interact).
*** ppt *** Polymerization of Fibrin
(OK, so once this polymerizes it seems like you should have a blood clot right.
After all polymers are quite hardy if we look at plastic which is a polymer
they are almost always strong. So what then could this Factor XIII be used
for?)
2. Role of Factor XIII
- Enforces the polymer
(Binding only temporary, need covalent bond: So at this point all that is
holding this polymer together is very strong binding interactions between
the remaining A-subunit and the -subunit. But as we know or should know,
binding is only a temporary thing. Something can bind an fall off quite easily.
In other words, it’s not a permanent link.)
(For a very strong polymer, one needs a permanent link or a covalent bond
to link together all of the monomers unit. So that is the job of Factor XIII is
to create covalent bonds that will permanently link the A-subunits and subunits)
- Creates covalent bonds between A-subunits/ -subunits
- Crosslinks Glu and Lys residues:
D. Hemophilia A
- No clotting, can bleed to death
- Result of two bad genes of Factor VIII
*** ppt *** Cause of Hemophilia A
*** ppt *** Treatment of Hemophilia A