exam 2 biochem practice questions Flashcards
what is the major themes in chapter 4
primary sequence to 3-D structure
structure = function
each protein has a unique structure (is also not static but dynamic)
non-covalent forces are very important
many structures have common patterns that are given a name (like domain and motif….I think)
what is different about protein compared to other organic polymers
what does the conformation of a protein allow the protein to do
has a specific 3-D conformation
allows the protein to function
what is a native fold
what can be said about its energy
the properly folded, functional conformation of a protein
it has the lowest free energy
what does the native fold have and are examples of it
The native fold has a large number of favorable interactions within the protein for stability
example: ”burying” hydrophobic groups and maximizing H-bonding
how is protein conformation stabilized by
what do these combat
Disulfide bonds
Weak, non-covalent interactions
THESE COMBAT ENTROPY
what are the Favorable Interactions in Protein Folding used to Maintain a Native State (fold)?
hydrophobic effect
hydrogen bonds
van der waals interactions
electrostatic interactions
what does the Hydrophobic Effect do for the native fold
Release of water molecules from the structured solvation layer around the molecule as protein folds increases the net entropy
Correctly position hydrophobic side chains depending on the environment
what does hydrogen bonding do for the native fold
Interaction of N-H and C=O of the peptide bond leads to local regular structures such as α-helices and β-sheets
- Side chain – side chain interactions. H-bonding between R-groups, H-bonding between backbone and R-groups.
what does van der waals do for the native fold
Weak attraction between all atoms contributes significantly to the stability in the interior of the protein
what do Electrostatic Interactions
dof ro native fold
Long-range strong interactions between permanently charged groups
Salt-bridges, especially buried in the hydrophobic environment strongly stabilize the protein
what does secondary structure start with
starts with primary structure
2 structures
describe the peptide bond
describe carbon-carbon bonds, what is another name for it
describe the carbon-nitrogen bond, what is another name for it
for psi/phi bonds, what can occur but what will be prevented
the peptide bond is rigid and planar, constraining the protein to certain, allowed conformations
Carbon-carbon (psi - Ψ) bonds can rotate
Carbon-nitrogen (peptide) bond (phi - Φ) cannot rotate
In theory, + / - 180° rotation for phi/psi bonds can occur,but…
- Steric Hinderance will prevent some angles from occurring…
what does steric hindrance effect and what is the result
steric hindrance can affect side chains and the backbone reduces possible rotations
what does a Ramachandran plot show
what does steric hindrance prevent
Shows common secondary structural elements and the acceptable range of rotation
Steric hindrance prevents all but a handful of secondarystructures
what is the structure of a peptide bond?
how many atoms does it have
is rigid and planar, constraining the protein to certain conformations
has 6 atoms
what do secondary structures consist of
alpha helix
beta-sheets
parallel beta sheets
antiparallel beta sheets
how is the helical backbone of an alpha helix held together?
what kind of direction is an alpha helix
what stabilizes the alpha helix
where are the side chains for the alpha helix
which residues align on top of each other
The helical backbone is held together by hydrogen bonds between the backbone amide of an “n” and carbonyl group of the “n + 4” amino acid
ann alpha helix is a right-handed helix
the hydrogen bond aligns roughly parallel with the helical axis and this stabilizes it
Side chains point out and are roughly perpendicular to the helical axis
residues 1 and 8 align on top of each other
describe the inner diameter of alpha helix, what can fit innside
describe the outer diameter of alpha helix, what can fit inside
what can amphipathic alpha helices form
what is the relatiionship between proline, glycines and alpha helices
does every polypeptide forrm a alpha helcies
what amino acids are strong helix formers
The inner diameter of the helix is about 4-5ÅToo small for anything to bind inside
The outer diameter of the helix (with side chains)is 10-12 Å. Fits into the major groove of dsDNA
Some amphipathic α-helices can form coiled-coildimers (stay tuned – keratin)
α-helices cannot be formed with Pro kink(disrupts the helix). Gly is a helix breaker too.
Not all polypeptide sequences adopt α-helical structures
Small hydrophobic residues such as Ala and Leu are stronghelix formers.
what is the cause of the sheet-like zig zag structure of beta sheets
what holds together the sheet-like arrangement for beta sheet
where are the side chains in beta sheets
what amino acids are found in beta sheets
what amino acids are not found in beta sheets
The planarity of the peptide bond and tetrahedral geometry of the α-carbon create a pleated sheet-like structure (zigzag)
Sheet-like arrangement of the backbone is held together by hydrogen bonds between the backbone amides and carbonyl groups in different strands
Side chains protrude from the sheet alternating in up and down directions
Found in β-Sheets:
Large, aromatic (Y, F, W)
Branched (T, V, I)
NOT Found in β-Sheets:
G, P
in parallel beta sheet, where do the H-Bonds run and what does it result in
where can the individual strands be in the primary structure
In parallel β-sheets, the H-bonded strands run in the same direction,
Resulting in bent H-bonds (weaker)
Individual strands can be close, or distant, in the primary structure
in anti-parallel beta sheet, where do the H-Bonds run and what does it result in
where can the individual strands be in the primary structure
In antiparallel β-sheets, the H-bonded strands run in opposite directions, Resulting in linear H-bonds (stronger)
Individual strands can be close, or distant, in the primary structure
how are most proteins shaped
what is the reason for this shape
how many A.A residues are in turns or loops
what do the arrows in strands and sometimes in helices indicate
when do beta turns occur
how is the 180* turn accomplished
how are beta turns stabilized
what amino acid is found in beta turn type 1
what amino acid is found in beta turn type 2
Most proteins are globular in shape
the globular shape is due to of frequent turns or loops in the polypeptide chain that connects beta strands and alpha-helices
1/3 of AA residues are in turns or loops
Strands, and sometimes helices, will have arrows to indicate N- and C- termini of the 2° structural element
β turns occur frequently whenever strands in β sheets change direction (so whenever the beta sheets turn to change direction)
The 180° turn is accomplished over 4 amino acids
Turn stabilized by a H-bond
type 1: proline
type 2: glycine
what is the tertiary structure and what does it include
how is it stabilized
what interactions largely make it up
how else can it be stabilized
what Is also possible
what are two major of tertiary structure
Tertiary Structure – overall 3D arrangement of all atoms in a protein
Includes long-range contacts between AA’s in a single polypeptide chain
‘Stabilized by numerous weak interactions between amino acid side chains
Largely hydrophobic and polar interactions
Can be stabilized by disulfide bonds
Side chain with backbone interactions are also possible
Two Major Groups
- Fibrous (elongated; structural)
- Globular (enzymes, etc.)
what do tertiary structures often have and is it solute in water
what does it contain a high proportion of and what are examples of these
are the underlying structures complicated
what does it have a high proportion of
what does it have extensive of
what are examples of fibrous proteins
Often have structural rather than dynamic roles and are water insoluble
Typically contain high proportions of α-helices (keratin) or β-pleated sheets (fibroin)
High proportion of hydrophobic AAs–that is why it is water insoluble!!!!!!!!!
Extensive supramolecular complexes
Fibrous Proteins: α-keratin, collagen, silk fibroin
where can you find alpha keratin
what is alpha keratin made of and what is ti strengthened by
what is the fact from legally blonde
how isa super tight coiled coil made
how is permanent waving of hair done
Hair, nails, hooves, horns, outer skin
made of Strong, RH α-helix; LH parallel super helix; strengthened by cross-links by covalent disulfide bonds – stabilizes!
perm removes disulfide bonds
Super-twisting helices, in a left-handed fashion, wrap around each other to make a very tight coiled-coil
permanent waving of hair: reduce disulfide bonds, moist heat breaks H-bonds and causes uncoiling of alpha heckles, remove reducing again, add oxidizing agent, new SS bonds
what does collagen provide and how many different types are there
where can collagen be found
how does each protein fold
what is the structure of collagen
what amino acids are found in collagen
which amino acid provides tensile strength for collagen
how many variants are there in mammals
Provides tensile strength and structure (~30 types of collagen).
can be found in Connective tissue (tendons, cartilage, organic matrix of bone, cornea)
Each protein folds into a left-handed helix (α-chains; NOT α-helix)
Coiled-coil of three separate α-chains supertwisted around each other in a right-handed manner to provide strength (like a rope).
find Gly, Ala, Pro, HyLys and HyPro (essential! specific to collagen)
Lys-HyLys (Hydroxylysine) form cross-links for added strength
30+ variants in mammals
in antiparallel beta sheets, where do H-bonds run
does this make the H-Bonds linear or bent and does it make them stronger or weaker?
what kind of amino acids is silk fibroin rich in
what kind of structure is it mostly made of
does it stretch
what kind of interactions are within
what kind of cross-links does it have, does it make it rigid or flexible
In antiparallel β-sheets, the H-bonded strands run in opposite directions
Resulting in linear H-bonds (stronger)
Individual strands can be close, or distant, in the primary structure
Rich in Ala and Gly
antiparallel β-sheets
Fully extended: no stretching
Extensive hydrogen bonding and van der Waals interactions between sheets, but no covalent cross-links for flexibility.
for silk fibroin
what is it used for today?
what are its characteristics?
what structure is it made of
what are its cross-links
used for silk
soft and flexible filaments
made of beta conformation
no cross-links
for collagen of tendons, bone matrix
how is it used today?
what are its characteristics?
what structure is it made of
what are its cross links
tendons, connective tissues, organic bone matrix
high tensile strength without stretch
made of collagen triple helix
– hydroxylysine (unique) cross-links structure = function
for alpha keratin
how is it used today?
what are its characteristics?
what structure is it made of
hair, feathers, and nails
tough, insoluble protective structures of varying hardness and flexibility
made of alpha helix, cross-linked by disulfide bonds
what are examples of globular proteins
what structures can be found in them
how are the different secondary structures arranged?
what does the folding provide
what is the general rule for globular proteins
examples: enzymes, transport proteins, molar proteins, regulatory proteins, immunoglobulins etc
alpha helices, beta sheets, beta turns can all be found
the different secondary structures are
- compact conformation
- folding provides structure diversity
general rule:
- bury nonpolar amino acid R-groups
- distant segments may come together, but not the norm
- optimize the number of weak interactions
what is an example of a globular protein?
what does this protein bind and where is it found
what is this protein related to but has a higher affinity for what
what are the rules followed for this protein?
what structures is this protein made of
how many amino acids is it made of
what kind of structure is it and what is at its center
what is its prosthetic group?
what is a prosthetic group?
what amino acid is attached to what molecule in its center
myoglobin!
binds iron and O2 and is found in muscles
related to hemoglobin but has a higher affinity for O2
general rules: the hydrophobic R groups are buried
made of alpha helices connected by loops
made of 153 amino acids
it is a porphyrin ring and has iron at its center
heme is it a prosthetic group
a prosthetic group is a nonprotein group forming part of or combined with a protein.
a proximal histidine group is attached to the iron, a distal histidine group hovers near the opposite face
what are
chymotrypsin
ribonuclease
carboxypeptidase
cytochrome C
lysosome
myoglobin
they are secondary structures!
what are the steps taken for X-ray crystallography?
Purify the protein
Crystallize the protein
Collect diffraction data (light bending)
Calculate electron density
Fit residues into density
what are the pros and cons of X-Ray crystallography
Pros:
- No size limits
- High resolution
- Well-established
Cons:
- Difficult for membrane proteins
- The crystal form may not represent the protein in solution or in the cell
- Not dynamic (cannot determine how the protein interacts with things)
what steps are taken for NMR
Steps
Grow the protein with a NMR-active isotope (usually of Carbon-13)
Purify the protein
Collect NMR data
Assign NMR signals
Calculate the structure
what are the pros and cons of NMR
Pros:
- No need to crystallize the protein
- Dynamic studies possible
- Interaction with ligands
Cons:
Difficult for insoluble proteins
Works best with small proteins
Nuclear Magnetic Resonance
what is a motif associated with
what are motifs
what are motifs composed of
what are what a motif is composed of also considered
motifs can be found as what
what are proteins made of
what are examples of secondary strcutures that can be found in motifs
Globular Proteins are associated with Motifs
Motifs are stable arrangements of several secondary structure elements
All alpha-helix
All beta-sheet
Combination
These are sometimes considered supersecondary structures
Motifs can be found as reoccurring structures in numerous proteins
Motifs can be found as reoccurring structures in numerous proteins
examples of secondary structures that can be found in motifs:
twisted beta-sheet
alpha alpha corner
beta alpha beta loop
beta barrel
what can you find in large motifs
what are the two classes that a motif can be organized into
You can find small motifs as a part of large motifs
People have determined the structure of so many proteins, so we must organize. Two classes of organization are shown here.
what are domains and where can they be found
what can a domain have
when do domains adopt the same folding pattern
what can a single protein have
can be found in globular proteins
Domains are relatively stable, independently folded regions within the tertiary structure of a globular protein
Each domain may encompass one or more motifs and have the same combination of motifs (EF-hand)
Domains having more than 30% of their amino acid sequence in common normally adopt the same folding pattern.
A single protein can have several domains with each domain performing a different function (e.g., enzymatic, docking, regulatory, pore, membrane anchoring, etc)
what cause a Quarternary (4°) Structure
how do the the polypeptide subunits associate into a larger functional cluster
What drives Quarternary Association?
Results from the association of two or more polypeptide subunits into a larger functional cluster
These polypeptide subunits associate into a larger functional cluster via side chain – side chain and side chain – polypeptide backbone interactions
what drives quaternary association:
Stability: reduction of surface to volume ratio
Genetic economy and efficiency (using quarternary structure makes our genome more efficient)
Bringing catalytic sites together
Cooperativity (biological function may be regulated by complex interactions of multiple subunits)
what do Intrinsically Disordered Proteins or Protein Segments
lack
what can this type of protein remain as or what can it also do
how many humans proteins fit this designation
what amino acids form then and
Contain protein segments that lack a definable structure. Or, the entire protein
The protein can remain in a primary structure indefinitely; or it may have a domain that can interact with other molecules, but the rest of the protein will remain disordered.
Possibly 1/3 of all human proteins fit this designation.
what is Proteostasis
how do proteins normally exiist
what is involved in mainatining proteostasis
Proteostasis is Protein Stability;Proteostasis is the constant level of the active set of proteins in a cell.
Proteins can exist in native conformation, or other conformations; flexibility is important
Maintaining proteostasis is a complex process, involving: synthesis, folding, unfolding, degradation, modification, etc
most protein fucntion depends on what
to be active what must a protein be
what can the order of amino acids determine for a protein
how can proteins denature
Most protein’s function depends on its 3D-structure.
To be active a protein must be correctly folded (native conformation)
The order of amino acids can help determine what this 3D conformation will be
Loss of structural integrity and function/activity is called denaturation
Proteins can be denatured by:
1. Strong acid or base
2. Organic solvents
3. Detergents
4. Reducing agents
5. Salt concentration
6. Heavy metal ions
7. Temperature
8. Mechanical stress
to what does a protein fold
what determines the direction of a protein
where can folding of 2ndary structure occur
how do secondary structures interact
when does protein synthesis being
Proteins fold to the lowest-energy (most stable) conformation in the microsecond to second-time scales.
It is not a “search”
The direction is biased towards thermodynamically and sterically possible conformations
Nonpolar inside, polar outside! (we’ve gone over this
Folding of secondary structures can occur at multiple sites at the same time.
Then, secondary structures interact to find the lowest energy minima (conformation)
Folding often begins before protein synthesis is complete!
what are chaperones
what is Hso70
what are chaperonins
What do Isomerases do?
another protein that promotes correct folding
Hsp70 (Heat Shock Protein 70) family protects unfolded proteins from denaturation and aggregation
Chaperonins promote correct folding
Isomerases make sure we have the correct stereochemistry
PDI – Protein disulfide isomerase
PPI – Peptide prolyl cis-trans isomerase
what if protein is misfolded
normally misfolded proteins are fixed (rendked) or degraded
defects n any of the ceeluar systems may affect the degree of protein misfolding
what is β-amyloid critical for and what shape must it be in
what happens in alzheimers
what does the misfolding promote
what is lost due to misfolding
then what happens to the brain
The native (correctly folded) β-amyloid is a soluble globular protein which is critical for neuronal growth, survival, and post-injury repair
In Alzheimer’s disease, bet amyloid is clipped from the cell membrane, fragmented, and then, it misfolds
This misfolding promotes aggregation
Correctly folded helices are lost, and peptides form β strands, β helices, and β sheets now insoluble
These insoluble plaques collect around the neurons and disrupt their environment and connectivity
what is Parkinson’s Disease, Lewy-Body Dementia
what is Huntington’s Disease
Parkinson’s Disease, Lewy-Body Dementia: misfolded α-synuclein forms aggregates Lewy Bodies
Huntington’s Disease: genetic mutation that increases CAG repeats (increases #of amino acids) causes misfolding and aggregation neuronal death
what is a ligand?
what is a binding site
what is protein flexibility important for
what else is important for binding
what are the two parts that an enzyme comes into contact with
what forces are responsible for binding and what does this allow for
a molecule that is reversiibily bound to a receptor
specific location on protein
protein flexibility is important for induced fit
regulation is important for binding
enzyme comes into contact with a substrate and catalytic (active) site
binding is via noncovalent forces that dictate protein structure which allows for interactions to be transient
what can the high specificity of ligand binding be explained by
what is Molecular complementarity due to
what is the lock and key model
are proteins rigid
what is the induced fit model
High specificity can be explained by the complementary nature of the binding site and the ligand
Molecular complementarity: size, shape, charge, hydrophobicity
Lock and key model: proteins and ligands have a rigid interaction with each other
Proteins are FLEXIBLE!
Induced Fit Model: both the ligand and theprotein can change conformations uponbinding; makes binding site more complementary to the ligand
what is one example of Illustrating Protein Function that we are focusing on
is O2 soluble
what can transport O2 but what is the consequence of it
because of the consequence, what do we need to do
how do we sequester Fe
what is a Heme a prosthetic group of
what is a prosthetic group
how does O2 bind
what does O2 bind
how manny sites does heme have and what binds those coordination sites
Illustrating Protein Function Through Oxygen Binding
O2 is critically important (obvs), but poorly soluble
Transition metals (esp. Fe) can transport O2 but can damage cells
So there is a need to sequester Fe
How? Heme!
Heme is a prosthetic group – a porphyrinring complex with an iron ion (Fe2+)
Binds oxygen reversibly
Oxygen binds via heme as amino acidscannot bind oxygen
6 coordination sites: 1-4 (nitrogen),5 (amino acid), 6 (oxygen)
what is myoglobin (Mb) and what is it composed of with how many amino acids
what is Mb mostly made of and what kind of regions does it have
what is its job
what does it contain
what interacts with heme and O2
what does it sterically inhibit O2 from doing
how is it folded and what is the result of this folding
Myoglobin is a compact globular protein composed of a single
polypeptide chain (153 amino acids in length)
Mainly α helices; there are 8. Also some intrinsically disordered regions (flexibility)
Carries and stores oxygen (poorly soluble)for muscles
Contains a heme prosthetic group
Histidines interact with heme and O2
Sterically inhibits oxygen from bindingperpendicularly to the heme plane (specific!)
Shaped/folded to form a “cradle” thatnestles the heme prosthetic group
—–Protects iron from oxidation (free radicals bad!)
in free heme, how does CO bind O2 compared to O2
In Mb, how does heme bind CO compared to O2
What does the protein structure act as
what is the effect of histidine on ligands
what does the histidine result in
Why not evolve Mb to bind O2 more preferentially and tightly relative to CO?
In free heme, carbon monoxide (CO) binds 20,000x better than O2
In Mb, heme binds CO only 40X better than O2
The protein structure acts as a gate.
The effect of histidine (His E7), forces ligands to bind at an angle.
Significantly improves O2 vs CO binding
what does Keq equal
what is the equation for the association constant and dissociation constant
what does it mean that if the Kd is high using the Eqn
what about if it is low using the Eqn
what s Ks relationship to Ka
Keq = [products]/[reactants]
Ka = [PL]/[P][L]
Kd = [P][L]/[PL]
high Kd means that there is more products which is [P] [L], so that means that the ligand would rather be dissociated from the receptor because t has a low affinity for for it
low Kd means that there is more reactants which is [PL], so that means that the ligand would rather be associated from the receptor because t has a high affinity for for it
use eqn of Kd to understand affinity!!!!!!!!!!!
Kd = 1/Ka
what increases the amount of O2 bound to Mb
what happens to the as the amount of O2 concentration increases sufficiently and what is the name for this
what does fraction saturation equal and what is the symbol and eqn
what determines that slope of the curve and what does it mean if it is very steep vs not as steep
the amount of O2 bound to Mb increases with O2 concentration
as the [O2] increases sufficiently, the amount of binding reaches a maximum value and this is called saturation
fraction saturation is the fraction (or half) of the protein that is bound to the ligand
Y or theta = [PL]/[Ptotal]
slope of the curve (how quickly we reach saturation) depends upon the affinity of the concentration
steep slope = high affinity
not so steep slope = low affinity
In a situation where P possesses one binding site for the ligand, what is the equation
what does it mean when the concentration of protein bound to ligand [PL] equals the concentration of free protein [P]
what does 50% saturation equal
what can we say about Kd = [L]
Kd = [P][L]/[PL] originally
Kd = [P]/[PL] ([L])
If the [PL] = [P] then [P]/[PL] = 1 and the concentration of free ligand [L] equals Kd
Kd = 1 [L]
For 50% saturation, Kd = L
Kd = [L] at which half of the available ligand-binding sites are occupied, so when P binds half of the L
what does theta equal
if [L] = 0 then theta equals
if [L]»_space; Kd then theta equals
f [L] «_space;Kd then theta equal
what happens to the curve if Kd gets smaller
theta is the fraction saturation which is
[PL] = [Ptotal] which is occupied binding sites over the total protein
eqn: theta = [L]/Kd + [L]
if [L] = 0 then theta equals 0
if [L]»_space; Kd then theta equals 1
f [L] «_space;Kd then theta equal 1/2
as the Kd gets smaller (binding is tigher) the binding curve is steepere
How do we know which of a series of candidate drugs is better than the others?
How do we know what dosage to give someone?
what is an agonist
what is an antagonist
Agonist – a compound that causes a physiological response
Antagonist – a compound that interferes with the physiological action of another compound
how is a gas concentration (hence ligand concentration) represented?
How well does Mb bind to O2 according to the binding curve?
looking into the binding curve, pO2 in the lungs is about 13 kPa, how well does O2 bind Mb accoridng to the curve
looking into the binding curve, pO2 in the lungs is about 4 kPa, how well does O2 bind Mb according to the curve
Is myoglobin a good transporter and deliverer of O2 in your body? Why?
What does myoglobin do?
is hemeglobin or Mb a better O2 transporter
as partial pressure so pO2
How well does myoglobin bind oxygen here?- very very well, indicated by the steep slope, so even a little bit of O2 is enough tot bind half of the ligands and then to saturate it to cause a plateau
at 13 kPa, there is a plateau which means that there is saturation and almost all of the ligands are bound
at 4 kPa, there is a forming plateau so there is almost saturation
Mb is not a good transporter because it has a very very hgh affinity for O2 so it does not want to let it go
Carries and stores oxygen (poorly soluble)for muscles
Our body prefers to use hemoglobin rather than myoglobin as the oxygen carrier in the bloodstream. This is because hemoglobin not only binds oxygen weakly but more importantly binds oxygen cooperatively.
Myoglobin (Mb) and Hemoglobin (Hb)
comparison
Mb
how many subunits
what does it do for O2
how many heme groups
Hb
how many subunits
what does it do for O2
how many heme groups
Myoglobin (Mb)
1 subunit
O2 storage
1 heme group
Hemoglobin (Hb)
4 subunits – 2α and 2β
O2 transport
4 heme groups
T state of Hb
what is called
when is it more stable
what affinity does it have O2
What is bound to it
is it rigid and if so what makes it rigid
what is a salt bridge and how are they broken
tense state
more stable in absence of O2
Lower affinity for O2
tense state
no O2 bound
salt bridges makes it rigid
salt bridges are ionic interactions between the R groups of the Hb
Salt bridges are broken when the O2 binds to the heme groups, makes it more relaxed
R state of Hb
what is called
when is it more stable
what affinity does it have O2
What is bound to it
what interacts
is it flexible and if so what makes it flexible
what is a salt bridge and how are they broken
Relaxed state
more stable in the presence of O2
higher affinity for O2
O2 bound
Beta subunits interact
salt bridges make it rigid
Salt bridges are broken when the O2 binds to the heme groups, makes it more relaxed
Relaxed state = fewer interactions, more flexible, higher affinity for O2
how many binding sites does Hb have for O2
What kind of protein iis Hb
what does that mean
what is cooperativity and what is it caused by
what is + cooperativity
what is - cooperativity
Recall that Hb has 4 O2 binding sites
Hb is an allosteric protein: having more than one conformation; binding at one site affects the affinity of another site)
This opens the door for something called cooperativity
With the binding of each O2 molecule, (which changes the conformation of the binding subunity from T to R), the affinity of the whole protein for O2 increases.
Positive Cooperativity: binding of a ligand increases the binding affinity of subsequent ligand
Negative Cooperativity: binding of a ligand decrases the binding affinity of the subsequent ligand
what does the Hb curve look likewhen we add cooperativity?
how does Hb bind O2
what does the curve look like for one binding site
how many binding sites does Mb have for O2 and that explains what
Mb can’t be cooperative. Why?
what does a sigmoidal curve represent
sigmoidal because Hb has a low affinity for O2
Hb binds O2 cooperatively
One binding site hyperbolic
Mb has only 1 binding site for O2 and this has a hyperbolic curve
Mb can’t be cooperative. Why? One binding site.
Hb has a sigmoidal curve. Reflects a transition from low-affinity to high-affinity binding. This makes Hb highly sensitive to changes in [O2].
physiologcally, how must Hb bind O2 and where and what is the kPa
physiologcally, where must Hb release O2and what is the kPa
what does effective transport entail and there what must happen
what is binding most sensitive to
what regulates Hb
what kind protein in Hb
Physiologically, Hb must bind O2 efficiently in the lungs (pO2 ~ 13.3 kPa)
Hb must release O2 in tissues (pO2 ~ 4 kPa)
Effective transport requires the ability to pick something up and drop if off.
Therefore, affinities must change.
Binding is much more sensitive to changes in oxygen concentration
Other ligands bind Hb and change its oxygen-binding properties Regulation!
Hb is ALLOSTERIC!
what does homotropic mean
what does heterotropic mean
what does Hb bind besides O2
Where does Hb bind O2 and CO2
what does binding CO2 or O2 result in
what can high [CO2] result in due to what process
What is the point of our circulation system
homotropic means the same ligands
heterotropic means different ligands
Hb binds CO2
Hb binds O2 & CO2 at different sites
binding one reduces the affinity for the other
high [CO2] raises the [H+]
in circulation:
- our RBCs have Hb and O2 from lungs
- the O2 binds to Hb
- Hb releases O2 to tissues
besides CO2 & O2, what does Hb bind
where is its binding site
what does the [CO2] influence
how is H+ formed
what happens when Hb binds H+
What is the main buffer system in RBCs
the affinity of Hb for O2 is what to the amount of what
what are the consequences for this what s the name for it!
Hb binds H+
Binds at different binding site from O2 & CO2
The [CO2] influences the [H+]: H+ is formed when CO2 reacts with Hb or H2O
CO2 + H2O <-> H2CO3 <-> H+ + HCO3
when Hb binds H+, its affinty for O2 decreases
Hb is the main buffer system in RBCs
the affinity of Hb for O2 is inversely proportional to amount of H+ & CO2 bound
consequences are the Bohr effect
- Peripheral tissues: high [CO2] and [H+]: low affinity
- lungs: low [CO2] & [H+]: high O2 affinity
what is BPG used for and does it stand for
what is the [O2] at high altitudes
where does BPG bind
what does it stabilize
what affinity does Hb have for O2 at T state
2,3-bisphosphoglycerate plays an important role in adaptation to high altitudes (low O2 partial pressure)
BPG binds to Hb at a site far from the O2 binding site
stabilizes the T state of Hb
(low affinity for O2
what happens to O2 at altitude
what does BPG regulate
does BPG share a binding ste
what kind of modulator is BPG
What does the stabilization of the T state mean for oxygen binding?
what varies at sea level
At altitude, since less oxygen is bound to Hb, that allows for BPG to do what
At altitude pO2 decreases
BPG regulates the affinity of Hb for O2
It has its own binding site
BPG is an allosteric modulator
Stabilization of the T state decreases the affinity of Hb for O2. So Hb is Less willing to bind and less willing to hold onto O2 so oxygen gets dropped off
The concentration of BPG varies in response to our altitude above sea level.
At altitude, since less oxygen is bound to Hb, that allows for BPG to fit into its binding site
Initially, at a lower pO2 (altitude), in the lungs, affinity of Hb for O2 is ______
what are the Kd values for when you are at lower altitude and what does this mean for the affinity compared to sea level
Within hours of being at altitude, the concentration of BPG ______ from __ mM to __ mM
what happened to the curve as BPG increased
What does BPG do to the binding curve?
Physiologically, what is happening?
Initially, at a lower pO2 (altitude), in the lungs, the affinity of Hb for O2 is reduced
at altitude, there is a higher Kd so O2 has a low affinity for Hb (or vice versa) and does not bind very well, Kd is 8mM, maybe because there is less O2
at sea level the Kd is 5mM which is less than 5 and says that at sea level there is a higher affinity for Kd
Within hours of being at altitude, the concentration of BPG increases from 5 mM to 8 mM
the curve is looking more hyperbolic showing that there is an increased affinity for O2, BPG brings that back up to sea level – levels.
What does BPG do to the binding curve?- Makes it more hyperbolic, BPG shifts the oxygen saturation curve to the right Hb has less affinity for O2 allowing it to release oxygen to the tissues (more than it did before)
Physiologically, what is happening?
- Hb is gaining less affinity for O2 so it can drop it off at the tissues
affinity in the lungs
Affinity for O2 in the lungs is reduced slightly, but in peripheral tissues more significantly; so, even though less is bound initially, more of it is released in the peripheral tissue; result: ~ 37% of bound O2 is delivered to peripheral tissue!
a study of the binding of hormone Z by receptor protein 1022, yielded the following data. what is the Kd for the binding of Z to P-1022
0.5 x 10^-9
1.0 x 10^-9
4.0 x 10^-9
10 x 10^-9
if [Protein] = [protein + ligand] then you can say y50% of L its bound
the answer is 4.0 x 10^-9 because. this is when the [protein] = [protein + ligand]
catalysts
Catalysts: change rate of reaction without a net change of itself. So: they increase reaction rates without being used up; do not alter equilibrium (Keq)
Enzyme
Vast majority of biological catalysts are what
how do globular proteins interact
a substance produced by a living organism that acts as a catalyst to bring about a specific biochemical reaction.
Vast majority of biological catalysts are globular proteins
globular protein interact via side-side chain interactions, h-bonding, ionic interactions and have subunits, domains and motifs
Substrate
Product
Active Site:
Substrate: substance acted upon (specific!)
Product: results
Active Site: location in the enzyme where thereaction occurs
what do enzymes do
wihtout enzymes, biochemical rxns are fast or slow?
Enzymes Increase Reaction Rates by lowering the activation energy
Without enzymes, biochemical reactions are slow. This is a good thing.
Enzymes can increase reaction rates (depending on the reaction) up to 1017 X faster than the uncatalyzed reaction!
what is the enzyme eqn
what do enzymes not effect
Interactions between enzyme and substrate at the active site occurs how
what is the rate limting step
E+ S <-> ES <-> transition state <->
EP <-> E + P
Many enzymes act in the forward and reverse reactions; helping the reaction reach equilibrium (but not affecting equilibrium, only rate)
Interactions between enzyme and substrate at the active site binding affinity
Enzyme activity is regulated by:
how many enzymes require metal ions
Holoenzyme
Coenzymes
Enzymes can be regulated
regulated by:
pH
Temperature
[enzyme]
[substrate]
[cofactors/coenzymes] (stay tuned)
1/3 of all known enzymes require metal ions(metalloenzymes)
Holoenzyme: apoenzyme (inactive) +cofactor/coenzyme/metal ion (prosthetic group)
Coenzymes are often vitamins and essential toour diet.
Enzymes can be regulated: enzymes can beactivated (phosphorylated) or inactivated (de-)
activation energy (Ea)
what is the free energy of activation ∆G‡
what does Ea equal in a graph
what does the rate of rxn relate to
A higher ∆G‡ corresponds to a fast or slow rxn
what is ∆G and is it changed by enzyme
the minimum amount of energy that colliding molecules have in order for a chemical rxn to take place
In biochemistry, activation energy is this is free energy of activation ∆G‡
on a graph, Ea is the difference between energy levels of the ground state and the transition state (typically higher energy than both ground states)
The rate of a reaction relates to activationenergy.
A higher ∆G‡ corresponds to a slowerreaction.
∆G is the energy difference between the substrateand product! NOT CHANGED BY ENZYME
enzymes increase rxn rates by
shifting equilibrium to the substrates
shifting equilibrium to the products
decreasing ∆G‡
increasing ∆G‡
decreasing ∆G‡
increasing ∆G‡
decreasing ∆G‡
what is not changed by an enzyme
what instead is changed by the enzyme
how are rxn rates set
what is the rate-limiting step
what does a catalyst do
what is ∆G‡ chemically
∆G (substrate and product) is not changed by an enzyme
The free energy difference between the substrate and the transition state ∆G‡ is changed by an enzyme
Reaction rates are set by the activation energy.
The activation energy “hill”, therefore, is the rate-limiting step!
A catalyst reduces the activation energy of a reaction!
∆G‡ is the amount of energy needed to convert 1 mol of the substrate from the ground state to the transition state
the binding site of an enzyme is most complementary to the
substrate
transition state
product
equilibrium
transition state
The more molecules reaching the transition state means what
what does the enzyme bind the best and what kind of interactions are there
The more molecules reaching the transition state means the more likely product forms, meaning the faster the reaction rate to reach equilibrium.
Enzymes bind the transition state BEST (more weak interactions). Induced fit andmolecular complementarity
enzyme-substrate complex
what is the enzyme-substrate complex
mediated by
what does induced fit lead to and what does it increase
Enzymes act as catalysts because of their ability to:
Enzyme-catalyzed reactions begin with the substrate enteringthe active site to form the enzyme-substrate complex
This is mediated by shape and weak, noncovalent interactions
Induced fit leads to both enzyme and substrate shape changes. This increases noncovalent interactions (desolvation - strip away H2O - Entropy?)
Enzymes act as catalysts because of their ability to:
- Bring substrate(s) and active sites together: proximity effect
- Hold substrate(s) at the exact distance and exact orientation necessary for the reaction: orientation effect
- Provide acidic, basic, or other types of groups required for catalysis: catalytic effect
- Lower the energy barrier by inducing strain in bonds in the substrate molecule: energy effect
How Enzymes Work: Catalytic Effect Mechanisms on their substrates
General acid-base catalysis
Covalent catalysis
Metal ion catalyst
General acid-base catalysis: proton donation/removal by an acid/base lowers free energy of the transition state
Covalent catalysis: acceleration through transient formation of an enzyme-substrate bond
Metal ion catalysis: metal ions
In reality, many enzymes use a combination of these strategies
participate in the reaction mechanism
Acid catalysis
Base catalysis
Amino Acids:
Acid catalysis: proton transfer from an acid lowers the free energy of the reaction’s transition state
Base catalysis: proton removal by a base lowers the free energy of the reaction’s transition state
Amino Acids: Glu, Asp, Lys, Arg, Cys, His, Ser, Tyr
metal ion + acid-base
Metal ions bound to the enzyme
Metal ions: help orient the substrate for the reaction
Stabilize negative charges
Mediate oxidation-reduction reactions
If we understand how enzymes catalyze reactions what can we do
Key Factors Affecting Rate:
Usually [E] «_space;[S]. Under these conditions…
If we understand how enzymes catalyze reactions, we can determine how to inhibit or stimulate them.
Key Factors Affecting Rate: concentrations of enzyme ([E]) and substrate(s) ([S])
The maximum achievable reaction rate is proportional to the concentration of the limiting reactant [E].
Michaelis and Menten developed a series of mathematical relationships to explain the behavior of many nonallosteric enzymes. what does nonallosteric mean
The initial reaction velocity (V0) is
The maximum velocity, (Vmax) is where
this process looks a lot like
nonallosteric means only 1 binding site
The initial reaction velocity (V0) is the rate at which substrate is consumed or product (molarity/time) is formed at the START of the reaction
The maximum velocity, (Vmax) is wherethe rate plateaus
looks like Kd
Note: Vmax is not an inherent propertyof an enzyme. It depends on reaction conditions [E]
At low [S], V0 increaseswith an increase in [S].
At higher [S], V0 increases by smaller and smaller amounts in response to increased [S].
The rate never exceeds a maximum rate, Vmax
What is Km
Small Km indicates
High Km indicates
Km is the
Km is a constant; it is intrinsic to the particular enzyme-substrate pair. For a given enzyme, each substrate has its own Km
Small Km indicates tight binding (curve shifted to the left)
High Km indicates weak binding (curve shifted to the right)
Km is the [S] at which half of the enzyme molecules have their active sites occupied with S and are generating ES.
Km is the [S] at which V0 = ½ Vmax
what do enzymes do
what does it do to the:
transition state
activation energy
rate constant (k)
delta G
promote the formation of a transition state
lower the activation energy
increase the magnitude of the rate constant for both the forward and reverse reactions, as the enzyme catalyzes both reactions.
cannot change the delta G (which is also equilibrium)
what terms are important for enzymatic reactions
[S]
Vo
Vmax
Km
what is Km
how does Km relate to Vmax
what is the Michaelis-Menten Equation
what does the graph look like
The substrate concentration at which half of the enzyme active sites are bound by substrate!
Km is the [S] that generates a rate of
1/2 Vmax
Vo = (Vmax)([S])/(Km + [S])
graph looks like a hyperbola
do we. always want some reactions to keep going? so what do we do
what can you do if you know a lot about the strcuture of an enzyme
how do many drugs work
Sometimes we want reactions to stop, so we inhibit enzymes
Alternatively, if you know a lot about the enzyme’s structure, one can rationally design an inhibitor to fit the active site (target-based design).
Many drugs, and toxic agents, act by inhibiting enzymes!
what are inhibitors
what do irreversible inhibitors do to an enzyme
what can one inhibitory molecule do
what are 2 examples of this
Irreversible Inhibitors (inactivators)
Usually cause stable, covalent alterations in the enzyme.
One inhibitory molecule can permanently shut off one enzyme molecule.
Often powerful toxins, but also drugs.
Example: organophosphorous compounds (sarin, insecticidesmalathion and parathion) bind to and inhibit acetylcholinesterase
Example:
aspirin binds to and inhibits cyclooxygenase (COX)
Example:
penicillin binds to and inhibits transpeptidase which makes bacterial cell walls
how do reversible inhibitors bind an enzyme?
how are they related to substrates?
they are used as drugs to do what
what are the 2 categories
Bind to, and dissociate from the enzyme (binding is easily reversed)
Interact with the enzyme through noncovalent association/dissociation reactions
They are often structural analogs of substrates (fit and inhibit) or products (feedback)
They are often used as drugs to slow down an enzyme
The fall into two major categories
- Competitive
- Not Competitive
Uncompetitive
Mixed
Reversible Enzyme Inhibition:
what can it bind to
what do they resemble
what do they do for the active site?
does it affect the catalysis for substrate to products, what does that mean for Vmax
how does increasing the [S] affect the type of inhibition, what does that mean for the Km
what kind of inhibitions is this
can bind
- the free enzyme and prevent binding of the substrate (Competitive)
- The enzyme-substrate complex and prevent the SP reaction
Competitive Inhibitors
- Resemble the normal substrate molecule
- Compete for admission into the active site
Inhibitor does not affect catalysis of SP, Vmax is unchanged!
Increasing the [S] favors the likelihood of S bindingto the enzyme instead of the inhibitor, I. Km shifts to the right on the graph and thus has a lower affinity
is competitive inhibition permanent
what is dihydrofolate
what is methotrexate
not permanent
Dihydrofolate is a substrate used for nucleotide synthesis
Cancer cells manufacture significant amounts of nucleotides necessary for rapid cell division
Methotrexate (inhibitor) is used for the treatment of some cancers (high doses)and for the treatment of various immunologic diseases such as rheumatoid arthritis
The Kinetics of Competitive Inhibition
what does increasing inhibition do to Km
even if we increase the inhibitor, does the Vmax change or stay the same
what is the inhibitor competing for
what happens if the [S] is really high
summary for competitive inhibtion
moves km to the right and lowers the affinity
Vmax stays the same
Compare the top blue curve (no inhibitor) with the other lines (increased [inhibitor])
Remember Km? Vmax?
Note: the presence of the inhibitor lowers andshifts the curve; affecting Km
All curves will eventually reach the sameplateau, and therefore the same Vmax
I is competing with S for the active site of E.
If [S] is really high, it will “win” the competition forthe active site
The Km has moved to the right in the presence of I, but Vmax is unchanged.
what does the lineweaver-Burk plot show
what is it the inverse of and what can it show us
Lineweaver-Burk (double-reciprocal) plots can be used to distinguish types of inhibition
Using the reciprocal of the Michaelis-Mententhe equation allows a more simplistic way to comparedifferent types of inhibition and easily extractkinetic values.
Competitive Inhibition
what does the y-intercept equal and does it change
what is the slope and does it change
what is the x-intercept and does it change
Competitive Inhibition: Increases Km; Vmax the same
Competitive Inhibition: Increases Km; Vmax the same
Pay attention to the slope… (Km/Vmax)
The y-intercept (1/Vmax) is always the same
The slope (Km/Vmax) increases with the inhibitor
The x-intercept (-1/Km) shifts to the right to showan increased Km in the presence of an inhibitor.
Reversible Enzyme Inhibition: Uncompetitive
what can reversible inhibitors bind
what does it bind to
how is its binding site created
how can inhibition be overcome
Reversible Inhibitors can bind to:
The free enzyme and prevents binding of the substrate (Competitive)
The enzyme-substrate complex and prevent the SP reaction (Uncompetitive)
Uncompetitive Inhibitors
- Binds only to the enzyme-substrate [ES] complex
The binding site for the inhibitor isn’t createduntil the enzyme binds the substrate
Inhibition cannot be overcome by the addition of moresubstrate
The Kinetics of Uncompetitive Inhibition
what does it bind
are any products formed?
do Km and Vmax increase, decrease or remain the same
what does it decrease
Compare the top blue curve (no inhibitor) with the other lines (increased [inhibitor])
In uncompetitive inhibition, the inhibitorbinds only to the ES complex. This E-S-I complexdoes not go to form any product.
Remember Vmax?
With a decreased Vmax, by definition, the Kmmust also decrease by the same amount as Vmax
Uncompetitive inhibition decreasesVmax and Km (though can be hard to see).
L/B Plots of Uncompetitive Inhibition
are Km and Vmax decreased, increased or remain the same
does the slope remain the same why or why not
does 1/Vmax (which is what) increase, decrease or remain the same
does the -1/Km shift right or left and what does that mean
on the plot, how are the lines in reference to each other
Uncompetitive Inhibition: Decreases Km and Vmax
Pay attention to the slope… (Km/Vmax)
The slope remains unchanged because Km and Vmax are reduced by equal amounts
The y-intercept (1/Vmax) decreases
The x-intercept (-1/Km) shifts to the left to showan decreased Km in the presence of inhibitor.
parallel lines!
in enzymology
what is the rate-limiting step
transition state
