Download Enzyme Regulation Strategies

9FUNDAMENTALS 1: 10:00 – 11:00
08/24/10
DELUCAS
ENZYME REGULATION STRATIGIES & PROTEOLYSIS
Scribe: Heather Thigpen
Proof: Tiffany Luke
Page 1 of 9
[ I.– XXIV. are slides from 8/23/10]
I.
II.
III.
IV.
V.
WHAT KIND OF COVALENT MODIFICATION REGULATE THE ACTIVITY OF ENZYMES [S29] - (From 8/23/10)
a. Lecture will cover covalent modifications in terms of a way to regulate enzymes & specific types of proteases
b. Phosphorylation is a reversible process
i. In biology, if we do something, we usually have to be able to undo it.
1. This is the case for regulatory enzymes
c. Phosphorylation is the prominent way in regulating enzymes
i. It involves a covalent modification in which a covalent bond is formed
ii. Generally, this phosphorylation is accomplish via protein kinases
1. There are hundreds of protein kinases in eukaryotic cells and a whole bunch in prokaryotic
d. Each protein kinase targets specific proteins for phosphorylation
e. If you put a phosphoryl group on something, you have to be able to remove it and clip that bond
i. A second enzyme, phosphoprotein phosphatase, catalyze the reverse reaction
f. Two key enzymes (protein kinase & phosphoprotein phosphatase) play critical role in regulation of enzymatic
reactions.
i. Since these enzymes play a role in regulation, they should also be regulated themselves– which they are.
WHAT KIND OF COVALENT MODIFICATION REGULATE THE ACTIVITY OF ENZYMES [S30]
a. There are 3 general types of protein kinases –
i. They target the residues of Serine (Ser), Threonine (Thr), & Tyrosine (Tyr).
1. So there are serine protein kinases, threonine protein kinases, etc.
b. The typical sequence that they recognize in the protein that they split is R(R/K)X(S/T)
i. Always have a R (First)
ii. Second one is maybe R =Arginine or K = Lysine
iii. Third one can be variable
iv. Last residue is either S = Serine or T = Threonine
c. Despite the fact that they are very specific – that each kinase recognizes a certain protein, the mechanism by
which they do this is very common.
d. Kinases are pretty large –about 260 residues make up the kinase core domain
e. One way that they regulate themselves is by intrasteric control
i. DeLucas states they never really talk about this term, but is used in the book
ii. One way to accomplish this is with a pseudosubstrate
1. If you have an area that looks similar to the substrate, but action can’t occur because some critical
amino acid is missing (like Ser or Thr). It will recognize it and will bind to it but won’t do anything.
2. The more you have of the pseudosubstrate, the kinase will be virtually ineffective.
3. This Is a way of “fooling” it & a way to regulate it
4. Key way to do this is replacing the Ser with a Alanine residue
a. Both small and about the same side so won’t sterically preclude its ability to interact with
substrate
5. Once we look at the reaction, we see that the –OH group of Serine is critical to form covalent bond to
form
a. Alanine doesn’t have –OH group
i. Can bind to but will not phosphorylate the substrate
WHAT KINDS OF COVALENT MODIFICATION REGULATE THE ACTIVITY OF ENZYMES [S31]
a. This table (also in book) points out the classes of kinases – Ser/Thr/Tyr, target sequences they recognize,
and activators of the process
i. There are activators & inhibitors
1. Activators turn on the kinases
b. Not expected to memorize
WHAT KIND OF COVALENT MODIFICATION REGULATE THE ACTIVITY OF ENZYMES [S32]
a. Skipped
CYCLIC AMP-DEPENDENT PROTEIN KINASE IS COMPOSED OF CATALYTIC AND REGULATORY SUBUNITS
[S33]
a. Cyclic AMP-dependent kinase is a tetramer – 4 subunits
i. Hetero-tetramer - the 2 C subunits are the same and the 2 R subunits are the same
1. Can be designated by α2β2
b. Have 2 catalytic subunits (C) & 2 regulatory subunits (R)
i. When cAMP binds to the 2 regulatory subunits
9FUNDAMENTALS 1: 10:00 – 11:00
Scribe: Heather Thigpen
08/24/10
Proof: Tiffany Luke
DELUCAS
ENZYME REGULATION STRATIGIES & PROTEOLYSIS
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1. R detaches from the C subunit and leaves the C subunit free to go phosphorylate proteins
ii. Why it doesn’t work like that is because it has a pseudo domain that is trying to be phosphorylated by the
catalytic subunit but because it has, say an Alanine, this can’t phosphorylate it
1. They sit there attracted to each other but it’s basically inhibited it. When cyclic AMP binds to
regulatory subunit, it creates a small conformational change that allows R to no longer have the right
configuration for C & R subunits to interact. They break apart so the new guy (C) will go do its work.
c. Using cyclic AMP kinase is just one way to regulate. There are many other ways.
VI. PHOSPHORYLATION IS NOT THE ONLY FORM OF COVALENT MODIFICATION THAT REGULATES PROTEIN
FUNCTION [S34]
a. There are other ways of covalent modifications, but DeLucas is not focusing on these
b. Might want to read over this section in the book that talks about modifying protein’s covalent bonds
i. Just so you know they are there and what some of them are & what they utilize in terms of ATP or UTP
c. Don’t have to memorize but should recognize that phosphorylation is only one of many ways to modify the
proteins.
VII. PHOSPHORYLATION IS NOT THE ONLY FORM OF COVALENT MODIFICATION THAT REGULATES PROTEIN
FUNCTION [S35]
a. Table (from book) where other covalent modifications are listed
i. Whether adenylation or methylation, etc., It is always where you get a covalent bond and can affect an
enzymes' ability to work better or not
b. Not going into detail – just know they exist
c. The kind of reaction is also shown in the table
VIII. ARE SOME ENZYMES CONTROLLED BY BOTH ALLOSTERIC REGULATION AND COVALENT
MODIFICATION? [S36]
a. Glycogen Phosphorylase is an example of an enzyme that’s regulated both ways we’ve talked about
i. Through hydrogen-bonding / an acid-base type reaction & covalent modification
1. Uses both of these in a specific ways depending on not only the level of substrates & other factors
that are involved, but also hormones in the body
a. This can cause you to completely forget about allosteric modifications & center on just the
covalent modifications
b. Glycogen Phosphorylase
i. Action of this enzyme is to cleave glucose from the non-reducing ends of glycogen
1. If you expend energy, you will eventually need to chew up your glycogen to create more energy/ATP
– this enzyme will help do that.
ii. Once it cleaves, it converts glycogen to glucose-1-phosphate
1. This is the first component of the reaction
2. This process involves a phosphorolysis reaction
a. Hydrolysis is when you use water to break bond, whereas phosphorolysis is when you use
phosphate to break the bond
c. Muscle Glycogen Phosphorylase is a dimer
i. Homo-dimer – both subunits are identical
1. Each subunit has a place where pyridoxal phosphate (PLP)/ inorganic phosphate binds
a. The concentration of PLP affects the ability of this enzyme to work
i. Cooperative type thing
ii. As phosphate binds, the reaction proceeds as a second order equation (opposed to
Michaelis-Menten equation) where you see a rapid rise & it’s easy for the second one to
bind.
d. There is an allosteric effector site where the subunits interact with each other
i. This allosteric site at the interface means something else binds there. When it binds, it will either make the
enzyme work better or worse.
IX. GP CONVERTS GLYCOGEN INTO READILY USABLE FUEL IN THE FORM OF GLUCOSE-1-PHOSPHATE [S37]
a. This shows the reaction as a phosphate group comes in; the phosphate group ends up being covalently
bound to the C-terminal glucose unit. The rest of the glycogen peptide unit is sitting there waiting for the next
one to come in and keep clipping these and creating glucose 1-phosphate.
X. PHOSPHOGLUCOMUTASE CONVERTS GLUCOSE-1-P INTO THE GLYCOLYTIC SUBSTRATE, GLUCOSE 6-P
[S38]
a. Glucose 6-phosphate is a derivative of glucose 1-phosphate
i. Shift phosphate from 1 to 6 position
b. Important because G 6-P is a precursor for the glycolysis reaction
i. This can be broken down to produce ATP
9FUNDAMENTALS 1: 10:00 – 11:00
Scribe: Heather Thigpen
08/24/10
Proof: Tiffany Luke
DELUCAS
ENZYME REGULATION STRATIGIES & PROTEOLYSIS
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c. Phosphoglucomutase – enzyme that allows the phosphate to shift from 1st position to 6th position
i. Mutase – type of enzyme that allows to shift from one position to another
XI. THE STRUCTURE OF GLYCOGEN PHOSPHORYLASE [S39]
a. It’s a dimer that’s 842 residue subunits
b. Each subunit contains an active site & allosteric effector site
i. 2 active sites in this enzyme, glycogen phosphorylase, & 2 allosteric sites in it
c. Has a Serine residue at position 14
1. Site for phosphorylation
d. The glycogen binding site, where the enzyme interacts with glycogen to start breaking it down, has regulatory
control.
e. There are several ways to regulate this enzyme (what the book is trying to point out)
i. Feedback, acceleration, or inhibition of the activity of this enzyme
f. Each subunit, although identical, are not covalently attached. There is a helical area that sticks out away from
the rest of it.
g. Imagine one big globular protein with this helical area sitting at the top and another helix comes together
upside down with it. The two helices interact to hold the structure together as a homo-dimer.
h. If that’s the case, what would you expect with those helices?
i. That there a lot of hydrophobic interactions on both sides of the interior of the helices so that they stick to
each other.
ii. On the outside there could be hydrophilic or hydrophobic, probably a mixture of interactions because of the
close contact with the globular domains.
iii. Would expect it to be more polar on the outside than on inside where the two helices are directly facing
each other
XII. THE STRUCTURE OF GLYCOGEN PHOSPHORYLASE [S40]
a. Drawing where barrels represents alpha helices
b. Imagine flipping the tower helices together to show how this structure would interact
c. Yellow – glycogen binding site
d. Allosteric site and PLP binding site are also shown
e. When looking at the picture, remember the other subunit will be on top of this with the helices connected
f. Point book makes is that these are 2 interacting subunits.
i. Why is this important?
1. This can be cooperative – meaning as the first one binds the second one would bind faster
2. So if the two subunits are interacting at this site, you can have a change in conformation or modified
conformation so when the first one binds, the second one would bind even better.
a. This is more than likely why they sit at that interface
3. Question from Student: When what binds?
a. {When an effector or inhibitor binds, it will change the conformation so that the other binding
sites will bind either faster or slower. This will be at the allosteric binding site since it is an
effector or inhibitor.} *
b. Actually, I said it wrong*, when this one binds it will change the conformation so that more of
the phosphate binds – which is responsible for the interaction
i. But it will also make it easier for the other one to bind, so you have this acceleration of
the reaction rate
c. Two things are actually held by the conformational change
XIII. GLYCOGEN PHOSPHORYLASE ACTIVITY IS REGULATED ALLOSTERICALLY [S41]
a. Muscle glycogen phosphorylase shows cooperativity in substrate binding
b. ATP & glucose-6-phosohate are allosteric inhibitors of glycogen phosphorylase
i. Remember as we break down glucose 1-P, it becomes G-6-P that enters into glycolysis and creates ATP.
1. Once you get enough energy/ATP, there needs to be something to shut this down
a. Reason why ATP and Glucose 6 Phosphate are inhibitors of this enzyme
c. The conformational change when ATP & G-6-P binds is different from when AMP binds.
i. AMP – only one phosphate ( as compared to ATP with 3)
ii. In an energy starved situation, there will be a buildup of AMP. There needs to be a way to tell the system
more energy is needed, so AMP binds to the same site( that ATP & G-6-P would) to cause a slightly
different conformational change.
1. Question from student: Which site? Allosteric effector site
9FUNDAMENTALS 1: 10:00 – 11:00
Scribe: Heather Thigpen
08/24/10
Proof: Tiffany Luke
DELUCAS
ENZYME REGULATION STRATIGIES & PROTEOLYSIS
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2. ATP, G-6-P, and AMP all bind to the same site. However, ATP & G-1-P cause a different response
than AMP. Allosterically, these cause a conformational change where an Aspartate gets replaced by
an Asparagine.
a. Asparagine has a positive charge (when trying to accelerate it)
b. Aspartate has a negative charge
c. You want to put a phosphate group in there, which is negatively charged, so if you change
this local environment so it is more conducive to a negative charge coming in then it will go
faster; therefore, getting more in there. This is exactly what is happening through this
allostery.
3. It would be just the opposite if you had the inhibitor AMP.
a. The negative Aspartate group is pushing into the site producing a conformational change
b. The Aspartate that was pointing out is now pointing in & makes it harder for the negatively
charged phosphate to come in and bind
d. When cell energy reserves are low, turn the system (glycogen stimulation) on.
i. high AMP & low ATP and G-6-P
e. When low AMP & high ATP and G-6-P, turn the system off
XIV.
GLYCOGEN PHOSPHORYLASE ACTIVITY IS REGULATED ALLOSTERICALLY [S42]
a. Velocity versus substrate concentration of these curves
b. If have AMP - Shift to the left
i. With less substrate (phosphate), will reach maximum velocity quicker
c. If have ATP - Shift to the right
i. Inhibit the reaction
XV. GLYCOGEN PHOSPHORYLASE CONFORMS TO THE MWC MODEL [S43]
*Dr. Whikehart sent out email explaining these models. Refer to that for better explanation*
a. Two models for allosteric – symmetry and causal models
i. This is the symmetry model
b. There are two conformations
i. Active conformation = R conformation
ii. Inactive conformation = T conformation
iii. R &T conformations exist in equilibrium
1. As something binds more to the R, the T shifts more to the R and causes the Aspartate to move and
the Asparagine to come in so that more phosphate can bind
iv. Question from student: Whether or not the T and the R are conformational changes?
1. In the causal model, when the substrate binds, it causes a comformational change so there are 2
conformations here and they are in equilibrium.
2. As the effector (whether AMP or others) binds to one, you have less without anything bound, you
have more of it shifts to that new conformation – that shifts that positive charge so that phosphate
can bind.
3. The allosteric control when something binds, it’s binding to the R state to activate. Not to the T state.
The part that it bound to has something else on it, more without it on it is created through the
equilibrium. So there is a shift, but not because of that binding.
4. AMP always binds to what little R state is there. These things are in equilibrium.
5. If you were in a starved state, & you now have some R state. AMP binds to it. Imagine that it takes
that out of that equation of equilibrium, so now that T state shifts to the R & more AMP bind. That is
the symmetry model & what’s happening here.
6. Yes, there is a conformational change, but only the effector binds to one state or the other. It doesn’t
make it change. That is the difference between causal and symmetry model.
v. Question from student: So you are saying there is a conformational change between T & R?
1. T & R are two different conformations & are in equilibrium.
a. If some R is taken away, more of T has to shift over.
b. Yes, there is a conformational change, but it is indirectly occurring.
i. Because when we bound an effector to R – it’s a different species now
ii. R without effector – you need more of it because of the equilibrium so more shift over –
that’s symmetry model
c. Yes, there is a conformational change but the point is the effector binds to the already
changed conformation
i. AMP binds to one conformation, G-6-P or ATP bind to another conformation
9FUNDAMENTALS 1: 10:00 – 11:00
Scribe: Heather Thigpen
08/24/10
Proof: Tiffany Luke
DELUCAS
ENZYME REGULATION STRATIGIES & PROTEOLYSIS
Page 5 of 9
ii. When they do, to create equilibrium again, that’s different – more has to shift over and
more of it will bind
d. Whereas with the causal model, it’s the same conformation.
i. AMP binds and causes one conformational change
ii. ATP binds to the very same conformation because the different change
e. It’s causing the change in that case – Casual Model
c. The conformational change at the interface is linked to the active sites
i. referred to this when talking about charges going in and out
XVI.
GLYCOGEN PHOSPHORYLASE IS CONTROLLED BY BOTH ALLOSTERIC REGULATION & COVALENT
MODIFICATION [S44]
a. Allosteric effect of amplifier or inhibitor causing to go to R state or go back to T state
i. Remember there is a Serine site that can be phosphorylated
1. When that happens, it shuts down this process & dominates the process
2. If phosphorylate at the Ser site – will shift things to the active state
a. Only way to deactivate this is with the phosphatase that clips that phosphate
b. This kind of allosteric regulation is occurring but on a smaller scale
i. Will not have much effect because the other process is dominating
XVII. A CONFROMATION CHANGE REGULATES ACTIVITY OF GLYCOGEN PHOSPHORYLASE [S45]
a. This is the full homo-dimer
i. Purple – tower helices
ii. When phosphates bind, causes a huge change in conformation
1. Shifts of 120 degrees
iii. In structures, difference between substrate bound and no substrate conformations are very different
1. There are places with loops that have certain degrees of freedom that allow them to flip and change
as much as they do
iv. This change in conformation is all due to phosphorylation of the Ser which is in red
v. When it is shifted like this, allosteric control is in a conformation where it doesn’t work well and therefore
shut down
1. This phosphorylation determines whether in the R state or T state
XVIII. REGULATION OF GP BY COVALENT MODIFICATION [S46]
a. Krebs & Fischer won Nobel prize for this phosphorylation occurs
XIX.
GLYCOGEN PHOSPHORYLASE IS ACTIVATED BY A CASCADE OF REACTIONS [S47]
a. Allosteric regulation only goes so far, if in need of ATP ( marathon running example), will have other ways of
phosphorylating to get this ATP
b. It all starts when hormones are released
i. These hormones will touch the outside of the inactive membrane protein, Adenyl cyclase causing a
conformational change
1. This will then activate the protein
ii. The active Adenyl cyclase will transform ATP into a cyclic structure – cAMP
1. cAMP is found in hundreds of reactions along membranes
a. it is a secondary messenger
i. when hormones or other proteins come up to a cell membrane and interact with some
receptor, often the message is carried inside the cell by cAMP to maybe even another
membrane protein or aqueous protein
2. This is the first process in getting to run a mile
iii. cAMP interacts with the inactivate cAMP-dependent protein kinase which creates the active form of cAMPdependent protein kinase
iv. Active cAMP dependent protein kinase will convert ATP to ADP which also converts the inactive
phosphorylase kinase to the active form.
v. The active phosphorylase kinase will take ATP to phosphorylate the serine residue on the inactive
glycogen phosphorylase b into active glycogen phosphorylase a
1. Active glycogen phosphorylase a is not acted on by much allosteric control
vi. Key is cAMP
XX. THE ADENYLYL CYCLASE REACTION [S48]
a. Skipped
XXI.
THE ADENYLYL CYCLASE REACTION [S49]
a. Skipped
XXII. CAMP IS A SECOND MESSENGER [S50]
a. Key is cAMP in the cascade
9FUNDAMENTALS 1: 10:00 – 11:00
Scribe: Heather Thigpen
08/24/10
Proof: Tiffany Luke
DELUCAS
ENZYME REGULATION STRATIGIES & PROTEOLYSIS
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b. Why is cascade so complicated?
i. All these are service controls, including hormone. There are several ways to control these.
c. Membrane protein that binds the hormone - called a G-protein coupled receptor.
i. It interacts with another protein on the inside of the cell called the G protein
d. G protein has 3 components – α, β & γ
i. Hetero-trimer
ii. When interacting, subunits are stable together
XXIII.
CAMP IS A SECOND MESSENGER [S51]
a. When hormone binds, GTP is released
i. GTP then binds to α subunit
1. GTP & α subunit will break away from other subunits
a. Swirly things in the diagram – have tail that can move through membrane to find the next
protein it wants to interact with to cause ATP to be dephosphorylated or to make cAMP
1. Occurs through G-protein coupled receptors
b. Process makes thousands of cAMP by one event
c. Reason cAMP is such an effective messenger (there’s a lot available)
d. Understand general concepts of how glycogen phosphorylase is regulated
e. END OF CHAPTER
XXIV. SHUTTLE PICTURE [S52] – (end of 8/23/10 slides)
a. Another story about space
XXV. PROTEOLYSIS [S1] - (8/24/10 slides)
a. Title Slide
XXVI. PROTEASES / PROTEOLYSIS [S2]
a. Proteases or peptidases are enzymes that catalyze the breakdown of peptide bonds in proteins/peptides
b. Proteolysis can be controlled or uncontrolled degradation of proteins/peptides
XXVII. NORMAL VS. NOT NORMAL [S3]
a. Normal – good things that they do with cells etc
b. Not Normal – types of diseases from abnormal functioning of proteases
i. Periodontal disease – proteases chew up tissue, allowing bacteria to spread
XXVIII. 14.5 WHAT CAN BE LEARNED FROM TYPICAL ENZYME MECHANISMS? [S4]
a. Serine proteases are classic example
i. Key to these are they have a catalytic triad
ii. Employs acid-base catalysis
XXIX. THE SERINE PROTEASES [S5]
a. Some typical Serine proteases: Trypsin. Chymotrypsin, elastase, thrombin and others (roles in red)
b. Factor D – get from urine & plays a role in alternate complement cascade
c. Involved in serine catalysis
d. Have a “catalytic triad” – 3 amino acids that are found in all serine proteases
i. Ser, His, Asp
e. Serine proteases are homologous, however locations of catalytic triad residues may differ somewhat
i. For convenience, always numbered His57, Asp102, and Ser195 (even if it’s not the actual location)
XXX. THE SERINE PROTEASES [S6]
a. The amino acid sequences of chymotrypsinogen, trypsin, and elastase
b. To stay on track with given numbers of catalytic triad, not all of the amino acids are counted because the
length is different
c. The filled in circles are were the amino acids are completely conserved
i. A lot of them tend to be around one of the three amino acids
d. The biological center of a protein, enzyme, etc. has more conservation
i. Important for a scaffold in structure, or in triad – the catalytic properties
XXXI. PROTEASE DISTRIBUTION [S7]
a. Skipped
XXXII. PROTEASES: MEMBERS OF THE SAME FAMILY ARE USUALLY EVOLUTIONARILY RELATED [S8]
a. There are other types of proteases other than serine proteases
i. Aspartic proteases
1. AIDS protease
2. Cathepsin D – linked to breast cancer
ii. Metalloproteases
iii. Cysteine proteases
9FUNDAMENTALS 1: 10:00 – 11:00
Scribe: Heather Thigpen
08/24/10
Proof: Tiffany Luke
DELUCAS
ENZYME REGULATION STRATIGIES & PROTEOLYSIS
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1. Has one that clips ubiquitine
2. Cruzipain – from organism T. Cruzi, important in life cycle of that organism which is a parasite
3. Sortase- found on many gram positive bacteria , used to break down tissue to evade it faster
b. The chemistry of how bonds are formed and broken in proteases
i. Cysteine proteases
1. Sulfur plays a key role
2. Carboxyl group of Asp helps orient the His
ii. Aspartic proteases
1. Has 2 Asp
2. Water plays key role in hydrolysis and sharing of atoms
a. Hydrogen is equally shared so it is a low bonding Hydrogen
i. It can move rapidly to create the reaction
iii. Metalloproteases
1. Have metal ion that helps to expel negative charges and accept electrons
XXXIII. PROTEASE CHART [S9]
a. Skipped
XXXIV. HOW DO ENZYMES WORK? HOW DOES ANY CATALYST WORK? [S10]
a. Skipped
XXXV. IONIZABLE AMINO ACIDS IN ENZYMES [S11]
a. Pointed out Nucleophile and Electrophile table & pKa values of amino acid table
XXXVI. CATALYTIC RESIDUES OF SERINE PROTEASES [S12]
a. The key 3 amino acids
b. Should read this to get a description of structures and how they work
XXXVII. DID CHYMOTRYPSIN EVOLVE VIA GENE DUPLICATION? [S13]
a. Chymotrypsin has 2 of the substituents, Asp, on one domain & Ser on other domain
i. If clipped, it is still active although less active
ii. Yes it probably did develop from gene duplication as a more effective way to speed up the reaction
because the way it orients the peptide along that way so it can clip it effectively
1. Has to do with having an oxyanion hole
a. When get to partial negative charge, need something to dispel that charge
i. Pocket from a Ser and a Gly help do that
2. Place for the main chain and substrate to bind
3. Specificity pocket – the amino acid next to where the clipping will occur
a. Will select out for specific amino acid
XXXVIII.
IDENTICAL FOLDS FOR SERINE PROTEASES [S14]
a. Picture just takes the main chains and sits them on top of each
b. Looks identical
c. The specificity pocket is what gives them specificity
XXXIX. CHYMOTRYPSIN STRUCTURE (PICTURE) [S15]
a. Skipped
XL. CONVERGENT EVOLUTION [S16]
a. Did (structure) come from convergent evolution?
i. If go back to bacteria, Subtilisin has triad like a serine protease
1. It did exist
XLI.
SERINE PROTEASE MECHANISM [S17]
a. Skipped
XLII.
4 IMPORTANT FEATURES [S18]
a. This is the key slide to look at
b. The Asp of catalytic triad helps stabilize the His in a planar configuration
c. Ser interacts with the N-H group of the His
d. Acid-base chemistry is also occurring
e. Carbon becomes tetrahedral
i. Electons shifted up to the oxygen
ii. In order stabilize this new tetrahedral arrangement from electronegativity, the same ser from the N group
that’s on the peptide bond not the R group has an H group that helps stabilize that negative charge.
1. H group also comes from a glycine
f. The actual peptide that will be cut interacts through its main chain
i. The C=O along the backbone interacts with N-H groups along the enzyme
9FUNDAMENTALS 1: 10:00 – 11:00
Scribe: Heather Thigpen
08/24/10
Proof: Tiffany Luke
DELUCAS
ENZYME REGULATION STRATIGIES & PROTEOLYSIS
Page 8 of 9
g. Some of these are clipped right on the carboxyl group of an aromatic group
h. The R group sits in the big hole
i. Depending on the R group, one protein might fit in there while another would be unable to clip there
i. The key is to allowing these different serine proteases to specify clipping next to an aliphatic group with a
negative charge at the end versus a hydrophobic group or something with a small R group (glycine)
XLIII. INHIBITOR BOUND ACTIVE SITES [S19]
a. Skipped
XLIV. CHYMOTRYPSIN (FREE ENZYME) – PICTURE [S20]
a. Skipped
XLV. ES COMPLEX – PICTURE [S21]
a. Don’t have time to go over but can easily follow the process step by step
b. Follow how this occurs, Don’t memorize
c. Should go through how it clips and how to get product
d.
THE “INTERMEDIATE” IS HIGHLY UNSTABLE. IT IS A CLOSE MIMIC OF THE TRANSITION STATE. [S22]
a. Skipped
XLVII. ACYL-ENZYME INTERMEDIATE – PICTURE [S23]
a. Skipped
XLVIII. ACYL-ENZYME INTERMEDIATE – PICTURE [S24]
a. Skipped
XLIX. SHORT-LIVED INTERMEDIATE – PICTURE [S25]
a. Skipped
L. ENZYME-PRODUCT 2 COMPLEX – PICTURE [S26]
a. Point out the specificity pocket
i. a aromatic group sits in this pocket
LI. THE CATALYTIC TRIAD OF THE SERINE PROTEASES [S27]
a. Skipped
LII. THE CATALYTIC TRIAD OF THE SERINE PROTEASES [S28]
a. Skipped
LIII. CHYMOTRYPSIN – TRYPSIN – ELASTASE – DIAGRAMS [S29]
a. How do these differentiate?
i. Chymotrypsin likes to clip on carboxyl side next to something that has a phenol group or big hydrophobic
group
1. Notice the specificity pocket for chymotrypsin
a. Has tiny amino acids there
b. Room for the phenyl group to sit in the pocket
ii. Trypsin cleaves on the side of a lysine
1. Lysine has a long chain with a positive charge
a. Has Asp group on the bottom of the pocket that are negatively charged
i. Will have room for long chain and interactions with Aspartate group
iii. Elastase cleaves next to amino acids that have very small R groups like Glycine
1. Specificity pocket is basically blocked
a. No room - why it can only cleave on carboxyl side next to small amino acid
b. Main point he wanted to get across – specificity pocket plays important role in where these proteases will
cleave
LIV.
SERINE PROTEASE BINDING POCKETS ARE ADAPTED TO PARTICULAR SUBSTRATES [S30]
a. Same thing that was talked about in S29
LV. SERINE PROTEASES CLEAVE ORGANIC ESTERS, SUCH AS p-NITROPHENYLACETATE [S31]
a. Skipped
LVI.
SERINE PROTEASES DISPLAY BURST KINETICS [S32]
a. Biphasic
i. Book describes it as burst kinetics
ii. Early phase – creating the first tetrahedral intermediate
1. Will break away a apart of a protein
a. Will need hydrolysis to do this
iii. Hydrolysis is a slower step
b. Can do burst kinetics with things that aren’t long proteins
i. Example in book para-Nitrophenolate
XLVI.
9FUNDAMENTALS 1: 10:00 – 11:00
Scribe: Heather Thigpen
08/24/10
Proof: Tiffany Luke
DELUCAS
ENZYME REGULATION STRATIGIES & PROTEOLYSIS
Page 9 of 9
LVII.
THE “OXYANIONHOLE” [S33]
a. Skipped
LVIII. TRANSITION-STATE STABILIZATION IN THE SERINE PROTEASES [S34]
a. Skipped
LIX.
ASPARTIC PROTEASES PLAY MANY ROLES IN HUMANS [S35] (end of lecture notes)
a. Table shows aspartic protease application and the fact they are involved in just two amino acids Asp – same
amino acid and water molecule
b. * Should know main things he talked about, don’t stress over the stuff that wasn’t really covered, and should
look over stuff he said to read in the book – the basics of it
[End 52:01 mins]