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Part 2


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2 types of oxygen binding proteins
3 oxygen binding structures that are paralogs:
# a.a. in Mb
# a.a. in hb a
# a.a. in hb B
2 ways to identify family resemblence among proteins:
primary structure (not as good)
tertiary structure (best)
homologous proteins
proteins that evolved from a
homologous (evolved from common ancestor) proteins that serve similar functions in different species.
homologous proteins that serve DIFFERENT function in same species.
Will an organism have one or more different paralogs of the same protein?
an organism can have many different paralogs of a particular ancestral protein.
statistical analysis of sequence alignments; Calculates the percentage of similar residues between the two.
Simplest method of SEQUENCE ALIGNMENT:
-slide the primary amino acid sequences past each other, one at a time.
-count the number of matched residues between the two proteins.
Scoring matrix
used to evaluate protein homology quantitatively after a sequence alignment.
why are sequence alignments useful?
we can construct evolutionary trees on the basis of relative divergence timelines. E.g., when b-Hb diverged from a-Hb, when a-Hb dvgd from Mb, and Mb from Leghemoglobin (the oldest).
# yrs ago Mb split from leghemoglobin
400 million yrs ago
at dawn of animal evolution
when did neanderthal man live?

When did anatomically modern man evolve?
400,000 to 30,000 years before now.

250,000 to 150,000 ybp

They coexisted!
Common ancestor of humans and neanderthals existed when?
paralogous or homologous proteins that are not surprising:
the GLOBINS: myoglobin, HbA and HbB.

Bovine Ribonuclease vs.
Human Ribonuclease
Paralogous Proteins that ARE surprising:
Angiogenin and Human Ribonuclease

Actin and Hsp-70
Human Ribonuclease
digestive enzyme
stimulates blood vessel growth

Heat shock protein 70
Function: assists folding proteins inside cells.

Actin - major cytoskeleton component.
Which binds O2 tighter; Mb or Hb?

what shape of binding curve do each have
Myglobin binds tighter;

Mb binding curve: HYPERBOLIC
Hb binding curve: SIGMOIDAL
Feature of Hb binding O2 that gives sigmoidal curve:
how does cooperative binding help Hb deliver more oxygen?
without cooperative binding, only 63% of binding sites would be full at 100torr in lungs. In tissues, 25% of binding sites still have O2; so only 38% of oxygen sites contribute to getting o2 to the body.
Which binding sites of Hb are weaker; first or last?
FIRST. Weakest.
What exacatly does O2 bind to on the Hb protein?
the Fe2+ on the heme ring.
normal oxidation state of IRON in the heme group under normal conditions:
FERROUS 2+ (two sites unbound - 1 in front, 1 in back)
What type of ring is the heme group?

Fe-protoporphryn IX
how many different polarities of sides are there on the heme ring?
3 sides are nonpolar
1 side is polar - has the two propionate side chains.
Which helices are situated arond the heme group, and how
E and F; at front and back.
between alpha helices e and f.
What happens when O2 binds to Fe2+?
Changes shape of the heme ring;
Fe2+ i too big to fit in the ring without oxygen; when oxygen binds, it goes into the plane of the ring.
Heterotropic regulation of Hb:
Hydrogen and CO2.

Both decrease Hb affinity for O2.
Hb releases O2 in tissues where pH is lower and CO2 conc. is higher. That's good cuz its what we want anyway.
Bohr effect
H and CO2 regulate release of O2 from Hemoglobin
Bohr's effect
H and CO2 help to stabilize deoxyhemoglobin. Favors release of O2. Stabilizes T state in tissues.
How many degrees do Alpha and beta dimers rotate when O2 binds to deoxyhemoglobin?
15 degrees
What happens when Deoxy (T) switches to Oxyhemoglobin (R)?
bonds between the dimers are broken.
how does H, Co2, and 2-3 BPG affect the binding curve of Hb?
Shifts it to the right.
At higher pressures of O2, lower conc. of O2 on binding sites.etc.
what is the sgnificance of 6 + charges in the center of DEOXY Hb?
2-3 BPG binds to 3 + charges on each beta chain of Hb. in teh central cavity of DEOXYHEMOGLOBIN.
Which HIS residue is the heme group bound to, what happens when O2 binds?
F8; it shifts and the interface btwn that subunit and the one that is on the other side of the interface w/ the F helix shifts. ionic bonds break.
What does 2-3 BPG do?
-Binds to deoxyhemoglobin in the central cavity to 6 + charges.
-Stabilizes deoxyhemoglobin.

Sickle cell anemia

Sickle cell trait
What procedure did Pauling use to compare HbS as SCTrait and SCAnemia with HbA? what were the results?

-HbS - a mutated form of HbA. Migrates toward the Cathode, because it's + chgd.
-HbS in trait gets 2 signals.
HbA was normal, by the Anode.
What's the difference in HbS from HbA?
-A Valine substituted for Glu in Beta subunits. HbS higher pI than HbA
-There are 2 beta subunits, so 2 less negative charges.
-Makes HbS more positive than HbA so it migrates toward the cathode.
What is catalysis a result of?
-Specifity for substrate
-allows for an alternate reaction pathway with a lower activation energy
What determines Keq?
G* = -2.3RTlog Keq
Specificity between an enzyme and substrate depends on...
Complementary fit for transition state
-either lock and key, or,
-induced fit
2 types of specifity of enzymes
Specific for Substrate
Specific for Reaction Mechanism
ES complex is what type of intermediate
binary complex
throughout the reaction, enzyme/substrate COMPLEX CONCENTRATION changes very LITTLE; essentialy stays the same.
2 ASSUMMPTIONS Michaelis-menten equation is based on:
That Enzyme only exists as Enzyme, and Enzyme-substrate.
That [S] is much greater than [E].
2 types of inhibitors

Action of irreversible inhibitor
-dissociates very slowly from enzyme.
-either covalent or noncovalent bind.
2 types of reversible inhibitors
Where do competitive inhibitors bind?
At the ACTIVE SITE - the same place as the substrate would have.

-Substrate can't bind then.
-so they bind to the active site.
How does competitive inhibition affect the RATE V
reduces the rate; not the same concent. of [ES]; some is [EI].
Noncompetitive Inhibition
Both substrate and inhibitor bind to the enzyme at different sites.
How does noncompetitive inhibition affect V the reaction rate?
It is decreased because when Enzymes are bound to substrates and inhibitors, they can't produce product.
How does a competitive inhibitor affect the kinetics of a reaction?
Vmax is not changed because Enzyme still binds to some S, while some binds to I. What is changed is Km because EI has to dissociate in order for there to be E to bind to S. So it affects the equilibrium, thus Km.
Can you overcome competitive inhibition? How?
YEs. by increasing the [S]
Can you overcome noncompetitive inhibition? How?
No. ESI forms; but it cannot produce product. So noncompetitive Inhibition essentially lowers the [E] that is functional.
How does noncompetitive inhibition alter the reaction kinetics?
Changes Vmax, but not Km. Less product is produced per time.
3 categories of IRREVERSIBLE INHIBITORS that help figure out the functionality of an active site
-group-specific reagents
-substrate analogs
-suicide inhibitors..
Group-specific reagents
Irreversible inhibitor that binds to specific R-groups of enzyme amino acid.

Ki - dissociation constant for COMPETITIVE inhibition
Ki = [E][I]/[EI]
Ki for NONCOMPETITIVE inhibitors

New apparent Km
Ki = [E][I]/[EI]
Ki'= [ES][I]/[ESI]

Kapp = Km(1 + [i]/Ki)
What changes will be seen on LBP for..

Competitive inhibition

Noncompetitive inhibition
COMP: Km is decreased. Vmax is same.

NONCOMP: Vmax is decreased. Km is same.
transition state analog
compounds resembling transition states of catalyzed reactions thare are
What happens during racemization of PROLINE?
-The alpha carbon of proline loses a proton.
-Transition state - negatively charged alpha carbon - TRIGONAL TRANS. STATE
-Reprotonation of alpha carbon in racemic conformation.
Pyrrole 2-Carboxylate
Transition state analog that mimics the trigonal transition state of proline racemization.
-its alpha carbon is trigonal, but has a double bond so its way more stable.
-binds way tighter.
Mixed non-competitive inhibitors
-Bind to ES, AND
-Bind to E

Both with different affinities, so affects both Vmax and Km.
Whats the distinct characteristic of Irreversible Inhibitors?
They often bind covalently - very strong bond - that modifies the enzyme.
-group-specific reagent.
-binds to the active SERINE of chymotrypsin.
What does DIPF do to Acetylcholinesterase?
Modifies Ser 195
Affinity label
molecule that is structurally similar to the SUBSTRATE that an enzyme is intended to catalyze.
-covalently modifies enzyme active site.
TPCK - what is it, what does it do.
-substrate analog of chymotrypsin.
-Irreversibly reacts with His 57 at the active site of chymotrypsin.
-Thus, it irreversibly inhibits and deactivates chymotrypsin.
(His 57 is part of the catalytic triad)
3 serine proteases to know
3 classes of proteases
-What do they do?
Serine Proteases
Aspartyl Proteases

-All cleave peptide bonds within proteins.
What is one Cysteine Protease?
What is one Aspartyl Protease?
What is one Metalloprotease?
3 Serine Proteases of similar structure
What type of inhibitors are transition state analogs?
COMPETITIVE - so reversible.
which doesnt make sense because they bind so titely.
Which 2 residues activeate Ser 195?
His and Asp
conditions for pseudo first order reaction rate of enzyme
[S] << Km

Very small amount of S so the increase in V is linearly proportional to [s]
Some examples of hydrolase enzyme actions.. what they do...
Digestive tract enzymes
blood clotting
cell signaling
cell death (apoptosis)
virus replication
Substrates Chymotrypsin is spcf for:
Tyrosine, Tryptophan, Phenylalanine, Methionine
Substrates Trypsin is specific for
Lysine and Arginine

-Large + charge molecules.
Structures Elastase is spcf for:
small uncharged a.a.
DIPF modifies:

TPCK modifies:
Ser 195

His 57
What are the 3 members of the catalytic triad??
Asp 102
His 57
Ser 195
What does the catalytic triad do?
Asp receives a H from His
His takes a H from Ser

Ser is more nucleophilic cuz its oxygen doesn't have the proton so close.
Ultimate accomplishment of catalytic triad (what happens to what type of charge)
Asp's - charge moves to Ser
After Ser 195 receives the negative charge from Asp 102, what does O on ser do?
The oxygen is electron rich, a nucleophile. It attacks the positively charged carbon of the peptide bond's C=O group.
Does only the reactive part of the peptide bind to chymotrypsin of the protease enzyme?
-No; several sites bind.

what is it
measure of substrate specificity.
-shows that large aromatic peptides are natural substrates for chymotrypsin.
Why does..
Chymotrypsin like aromatic Rgroups?
Trypsin like Lys, Arg?
Elastase like small uncharged?
What does the S1 pocket do?
binds an appropriate side chain of a peptide chain into the pocket and positions the adjacent peptide bond in the active site for cleavage.
What is the main difference between the serine proteases?
S1 pockets.
Chymotrypsin pocket is deep, with no charge.
Trypsin is deep, with Asp - negative charge binds to positive Lys and Arg.
Elastase is shallow with two valines blocking large R-chains.
Whats the purpose of the oxyanion hole?
Stabilizes the tetrahedral transiiton state during serine protease hydrolysis of a peptide bond.
(oxygen is negatively charged after being attacked)
an amino acid in the oxyanion hole is...
Gly 193
His 57's role in the mecanism of peptide hydrolysis by chymotrypsin:
First a proton ACCEPTOR (from Ser OH)
Then a proton DONOR

-In both steps.
Don't forget the amine formed, and the molecule of water added in step 2
how does a peptide initially bind to chymotrypsin?
H-bonding between enzyme and substrate.
what are the strategies of serine protease catalysis?
-Nucleophilic attack by Ser 195
-Covalent catalysis (acylenzyme forms)
-oxyanion transition state stabilizing
-general acid base proton movement by His 57
acyl enzyme
the intermediate of serine protease hydrolysis of protein peptide bonds after the amine is lost.
aspartyl proteases
-how do they hydrolize peptide bonds?
-what is active site structure?
each half of the protein has an aspartate group so that together they activate a water molecule and hydrolyze the peptide bond.
-One side accepts proton from water to make it a better nucleophile.
-other side donates proton to carbonyl oxygen of peptide to make it more susceptible to attack.
an aspartyl protease
The functions of each aspartate of aspartyl proteases
Left side: accepts proton to activate water

Right side: donates proton to stabilize oxyanion.
Aspartyl proteases have a tetrahedral transition state too!
just know it
-what does it do
Cleaves viral proteins into their active form. Makes the virus proliferate. A BAD THING.

want to prevent it.
What is unique about HIV proteases?
They are like aspartyl proteases, but are DEFINETELY IDENTICAL halves. Symmetric.

-Cleaves polypeptides into active forms essential for virus replication - yikes
-competitive inhibitor (binds to the actual site where HIV protein would)
-transition state analog
-binds very tightly to HIV Protease so that it can't cleave other virus peptides and activate them.
What does crixivan look like?
the HIV protein peptides that HIV protease would normally cleave in order to activate the virus for replication.

-A trans. state analog that prevents HIV replication - a good thing!
inactive precursors to pancreatic enzymes like chymotrypsin trypsin elastase carboxypeptidase and pepsin.
inactive precursor to chymotrypsin.
-synthesized in the pancreas.
-gets cleaved by TRYPSIN to give pi-chymotrypsin. THIS IS FULLY ACTIVE.
-pi-chymotrypsin cleaves another molecule of itself. gives alpha.
-alpha-chymotrypsin is cut into 3 pieces. the pieces stay close tho.
STRIKING feature of chymotrypsinogen activation
cleavage of ONE specific peptide bond by trypsin results in a fully active enzyme, chymotrypsin.
What happens to the oxyanion hole in activation of chymotrypsin from chymotrypsinogen?
a necessary NH group for stabilizing the tetrahedral C-O in protolysis is not there in chymotrypsinogen, but is moved into the correct conformation in chymotrypsin. Allows the enzyme to be ACTIVE!!!
bOND BETWEEN +charged Ile 16 and -charged Asp 194. Allows residues 193 and 195 to hydrogen bond with tetrahedral trans. state of peptide being cleaved.
how many protease activations does trypsin trigger when it gets activated itself?
how is blood clotting initiated?
by a serine protease cascade.
what are the two pathways to initiate the serine protease cascade for blood clotting?
What is FIBRIN
How is it formed
What is it for?
Fibrin is a protein that forms clots.

-It's converted from fibrinogen by thrombin. (a proteolytic enzyme)
-It's for forming clots duh
a proteolytic enzyme that cleaves fibrinogen into fibrin to form clots.
special case: when k-1 is much greater than k2 what happens
ES complex breaks down into E + S much more than product is produced. so,

Km=Kes; the Km is equal to the dissociation constant of the ES complex, and equals k-1/k1. because the k2 cancels out of the equation.
When k-1 is much greater than k2, Km is a measure of
the strength of the ES complex. If it's LARGE, the complex is weakly bound.
If it's small, it does not dissociate easily so bound strong.
The number of substrate molecules turned into product in a unit of time, when all the enzyme active sites are full.
What is significance of Kcat/Km
In normal cells, the [S] is not normally much greater than Km. so,

-characterizes the kinetics of an enzyme under more typical cellular conditions.
a rate constant for the reaction of E and S under typical cellular conditions.
-Measures enzyme preference for different substrates.
What is the limit of an enzyme's rate in cellular conditions? the limit of kcat/Km, the rate constant?
Even if the reaction of ES to Product (k2) is extremely fast, faster than the rate of ES breakdown (k-1), it cannot be faster than the rate of ES formation (k1) according to the laws of diffusion. Meaning, not > than 10^9

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