Midterm

Contains Modules 1-5

What are the functions of proteins?

* structural components of the cell
* sensors for environmental changes and mechanisms for relying this information to the cell
* enzymes, catalysts for chemical reactions
* gene regulation
* molecular motors
* signalling molecules between cells
* organelle identity and function
* proteins do everything
* shape and structure = function

What are proteins composed of?

* amino acids are attached in a linear array to create the primary structure of the protein = a polypeptide chain
* 20 different amino acids are incorporated into newly synthesized polypeptide

What is an amino acid?

* has a variable R-group (or side chain) that determines the properties of the side chains (R) that affect protein structure and function
* all have the same general structure of a carboxyl group, an amino group, and the variable R-group

What are the side chains of the amino acids?

* the side chains differ in: size, shape, charge, hydrophobicity, reactivity
* these properties can all have an effect on the confirmation of the whole protein
* how the side chains react indicated how the protein folds
* amino acids are classified into groups based on solubility in water or polarity of the side chain

What is solubility in variable side chains?

* solubility refers to a physical property of a molecule that can transiently bond with water through hydrogen bonding
* this is thermodynamically favourable

What is a hydrophilic molecule?

* (or portion of a molecule) is one that is typically charge-polarized and capable of hydrogen bonding with water

What is a hydrophobic molecule?

* not electrically polarized and unable to form hydrogen bonds, thus water repels them in favour of bonding with itself

What sorts of molecules are hydrophobic?

* anything unable to form hydrogen bonds (oils, fats, saturated hydrocarbons; a long chain of carbon linked together by single bonds)
* this can extend our side chain residues
* the general formula is CnH2n + 2 = alkane
* the simplest alkane is methane, CH4
* a protein in a hydrophobic environment will have the opposite structure (hydrophobic amino acids accumulating in the exterior of the protein)

What sorts of molecules are water insoluble (or only slightly soluble) amino acids?

* hydrophobic amino acids tend to be in the interior (core) of soluble proteins
* interior of cytosolic protein and form a hydrophobic core

What are the hydrophobic amino acids?

* non-polar side chains
* aromatic amino acids:
* Phenylalanine
* Tyrosine
* Tryotophan
* aliphatic amino acids (hydrocarbon chains)
* Alanine
* Valine
* Isoleucine
* Leucine
* Methionine

What sorts of molecules are water soluble?

* a hydrophilic molecule or portion of a molecule is one that is typically charged and capable of hydrogen bonding (on the outside of the protein)
* charged (ionized) at pH 7.0 physiologically)
* molecules with an -OH at one end (O-)
* molecules with an -NH2 at one and (NH3+)
* tend to be on the surface of the proteins
* make proteins soluble in aqueous solutions

What are the hydrophilic amino acids?

* they are typically charged
* basic amino acids (positively charged):
* Lysine
* Arginine
* acidic amino acids (negatively charged)
* aspartic acid
* glutamic acid
* these four amino acids are the prime contributors to the overall charge (aka domain) of the protein

What are the hydrophilic amino acids that are polar and uncharged?

* Serine and Threonine are uncharged at neutral pH but have polar -OH groups that participate in hydrogen bonds
* Asparagine and Glutamine are uncharged but have polar amide groups

What are the special amino acids?

* Cysteine → disulphide bridges formed with other cysteine molecules
* Glycine → the smallest amino acid, and it allows many bends
* Proline → forces a kink in the peptide chain which is essential for structure
* Histidine → has a pH dependent charge and can be negative or positive

How do the amino acids bind together?

* amino acids can be covalently bonded together into peptides by the formation of a peptide bond (condensation reaction releases a molecule of water)
* peptide bonds are formed by a condensation reaction between amino group of one amino acid and the carbonyl group of another

What is a peptide chain?

* a protein chain is conventionally depicted with its amino terminal on the left and its carboxyl terminal on the right

What is the primary structure?

* the linear arrangement (sequence) of amino acids
* the amino acid sequence is determined by the nucleotide sequence of the ending gene
* number of different polypeptide sequences? 20^n
* infinite number of sequences that could be made for amino acids

What are the different ways to fold the polypeptide?

* polypeptides spontaneously assume a random coil structure
* local interactions stabilize periodically ordered structures, so the term statistical coil is often used
* ionic bonds
* hydrogen bonds
* Van der Waal forces
* hydrophobic effect → noncovalent interactions, weak attractive forces (in an aggregate becomes very strong)

What is an ionic bond?

* attraction between a positively charged cation and negatively charged anion
* assisting in holding together the protein shape

What is a hydrogen bond?

* interaction between a partially positively-charged hydrogen atom in a molecular dipole and unpaired electrons from another atom

What is the hydrophobic effect?

* aggregation of non-polar molecule (side chains) in an aqueous medium in order to reduce the number of interaction with water
* a reduction in the hydrophobic surface area exposed to water (e.g. an oil droplet in water)
* the high energy (high entropy) state in the right is more energetically favourable than the more ordered state in the left

What are Van der Waal/London Dispersion Forces?

* weak non-specific attractive forces
* results from the creation of a transient dipole when two non-covalently bonded atoms are close enough together to perturb the distribution of electrons in one another (transient dipole is created) → only when dipoles are close together
* responsible for the association of non-polar molecules that cannot form hydrogen bonds or ionic bonds

What is the effect of more Van der Waal interactions between the amino acids?

* this creates a stronger association
* these non-covalent interactions are all weak attractive forces
* individually, they cannot maintain protein structures
* the accumulated effect of many bonds - therefore, the positions of interacting molecules must correspond to maximize the number of interactions
* the more interactions, the stronger the overall association

What is secondary structure?

* conformation of a portion of the polypeptide

What are motifs?

* combinations of secondary structures
* built from particular combinations of secondary structures
* structural units that recur in a variety of proteins
* exhibit particular 3-D architecture
* usually associated with a particular function

What is an alpha-helix?

* spiral, rod-like structures

What are beta-pleated sheets?

* planar structure, composed of alignments of 2 or more strands

What are turns/loops?

* connectors (bends in the protein shapes)

What are the characteristics of a regular, spiral conformation of an alpha-helix?

* carbonyl oxygen of each peptide bond is H-bonded to the amine hydrogen of the amino acid four residues towards the C-terminus
* forms independently of specific amino acid side chains

What type of secondary structure is this? List its properties?

* a cylinder with side chains pointing out
* R-groups determine hydrophobic/hydrophilic quality of the outer surface of the helix
* R groups determine the qualities
* diameter of the structure indicates strength

What are the characteristics of a beta-pleated sheet?

* laterally packed beta-strands (5 to 8 amino acid residues long)
* H-bonds between carbonyl and amino groups of backbone in adjacent beta strands
* side chains at the top and the bottom
* R-groups determine hydrophobic/hydrophilic quality of the surfaces of the sheets

What is a Beta Turn?

* involves 3 or 4 acid residues and is often found connecting the strands of beta sheets

What is a coiled-coil motif?

* hydrophobic surface
* creates a coil
* leucine zipper

What is a zinc-finger motif?

* alternate variants:
* C2H2 zinc finger
* C4 zinc finger
* C6 zinc finger
* alpha helix and two beta strands
* often in DNA binding
* consists of an alpha-helix and 2 beta strands, held in position by the interaction of precisely positioned Cys (C) or His (H) residues with a zinc atom

What is a Beta-Barrel motif?

* a beta-sheet forms a barrel when the last beta strand forms hydrogen bonds with the first strand
* first and last sheet create H-bonds
* R-groups at interior are hydrophilic

What is the Helix-Loop-Helix Motif?

* two alpha-helices joined by a loop region
* loop region can bind Ca2+ (co-factor) via carboxyl side chains from Asp or Glu in the loop
* protein structure and function depend on the co-factor

What is the tertiary structure?

* three dimensional arrangement of all amino acid residues of a single polypeptide
* the overall conformation of a single polypeptide
* fundamental unit of tertiary structure of a protein of the domain

What is a domain?

* a substructure produced by any part of a polypeptide chain that can fold independently into a compact, stable structure

What are functional domain?

* regions of a protein that perform a certain activity
* e.g. DNA binding, enzymatic, protein-protein interaction

What is a structural domain?

* regions of the protein that form compact, largely independent globular domains
* e.g. proline-rich, acidic domain

What is quaternary structure?

* number and organization of subunits in a multimeric protein

What is a multi-metric protein?

* a functional protein composed of multiple polypeptides

What is a dimer?

* two polypeptides or two subunits

What is a trimer?

* three polypeptides

What is a homodimer?

* two identical polymers

What is a heterodimer?

* two different polypeptides

What are unstructured proteins?

* lack a tertiary structure as isolated subunits

How do proteins acquire structure?

* DNA associated with zinc-finger complexes
* must establish a substrate

What are post-translational modifications?

* probably every protein in the cell is chemically modified after synthesis
* chemical modifications of individual amino acid side chains alter protein folding or localization and therefore regulate the function of the protein

What is acetylation?

* protects intracellular protease degradation (80% of proteins)
* addition of the acetyl group
* modifies activity

What is methylation?

* histodine residues are commonly modified to form 3-methyl histodine
* proteins at histodine residues also alters gene expression
* can weaken the association between histone and DNA
* reversible

What is phosphorylation?

* transfer of a phosphate group from ATP to the -OH group of serine, tyrosine, or threonine by kinases
* removal of phosphate group by phosphatase
* can activate and deactivate proteins by changing the ability of the protein to interact with a substrate

What is hydroxylation?

* addition of hydroxyl groups (OH)
* triple-helical coiled-coil of collagen (needs 3 of these)

What is carboxylation?

* addition of carboxyl groups (COO-)
* adding a negative charge
* can facilitate ion bind formation or allow charged cofactors to bind

What is glycosylation?

* addition of carbohydrates
* sugars added to -OH groups of serine and threonine (occurs in Golgi apparatus)
* protects proteins from proteolysis and for proper protein folding

What is lipidation?

* addition of lipid molecules
* anchors proteins to membranes

What is the assembly of the native conformation?

* the native state is the most thermodynamically stable confirmation of a protein
* very difficult to guess tertiary structure
* protein folding is:
* easy
* spontaneous
* reversible
* unique

What is the reversible denaturation experiment?

* renaturation of protein in vitro occurs spontaneously
* the information for folding a protein lies in its sequence
* dialysis removes the denaturants

What are the three steps of correcting mis-folding of proteins?

1. most protein molecules fold rapidly into their correct configuration
2. incompletely folded proteins are helped to fold by chaperone proteins
3. mis-folded proteins are recognized for degradation

What is the general principle of protein folding?

* to prevent inappropriate interactions between amino acid residues and increase the efficiency of protein folding

What are the two types of chaperones?

* monomeric molecular chaperones
* multimeric chaperonin complexes
* not specific to a subset of proteins, but can assist many different proteins with distinct structures and functions

What are heat shock proteins (Hsp)?

* Hsp 70 in cytosol and mitochondria
* BiP in endoplasmic reticulum
* DnaK in bacteria
* high levels under stress like heat (cell responds to refold proteins) so they do not sustain damage
* bind to hydrophobic R groups and prevent and nascent polypeptide from associating with other proteins or from folding prematurely, and from aggregating with other hydrophobic residues

What is the Hsp 70 family of heat-shock proteins?

* hydrophobic proteins on unfolded proteins quickly bind to hydrophobic residues on Hsp 70
* ATP hydrolysis to ADP changes the confirmation of the Hsp 70 chaperone
* this concomitantly changes the relative shape of the target protein, allowing it to fold properly
* ATP hydrolysis is stimulated by the co-chaperone DnaJ or Hsp 70
* ADP is released from Hsp 70, assisted by the nucleotide exchange factor GrpE or BAG1

What do chaperones form?

* they form folding chambers
* The chaperonin complex consists of two large subunits.
* Multiple proteins form the walls of the two GroEL or large subunits that are attached to one another at their bases.
* Each of the tops of the chambers are alternately open or capped by a GroES or small subunit.
* The cross‐section at the bottom illustrates that the GroEL is a hollow chamber that forms an isolation chamber or folding chamber

How does folding occur within the chaperones?

* At step one we are seeing the bottom chamber releasing the GroES cap and ADP as it completes its activity and the top chamber binding to ATP and a new substrate peptide
* A new GroES cap binds to the top of the GroEL, closing the chamber and isolating substrate peptide.
* A conformational change enlarges the chamber dimensions, giving the target peptide room to fold.
* The chamber remains closed for several seconds as the protein, in isolation, is allowed to fold.
* The chaperonin is not folding the protein directly, but allowing it to fold within the confines of the chamber.
* ATP hydrolysis allows the GroES cap to come off and the protein diffuses out.
* Hopefully it has been able to fold, but if not, it may go through this process again.
* Next time, the chaperonin will use the bottom chamber once again

What are the GROEL subunits?

* The walls of the GroEL chambers are made up of proteins called Hsp60.
* One Hsp60 protein is shown in a ribbon diagram.
* Three distinct domains are labelled:
* the apical domain at the chamber rim
* the equatorial domain at the bottom of the chamber or middle of the chaperonin complex
* the intermediate or hinge domain in the middle.
* In the top view of the GroEL, the 7 Hsp60 subunits that form the wall of a single GroEL can be seen.
* Each Hsp60 binds to a single ATP molecule so 7 ATP molecules are required for GroEL activity at any one time

How does the Hsp60 protein change shape?

* it is the individual behaviour of the subunits that lead to the overall changes

What types of proteins are degraded?

* mis-folded proteins
* denatured proteins
* proteins at too high a concentration
* proteins taken up into the cell
* regulated proteins
* these agrigates can be dangerous

What are the two steps of protein degradation

1. tagging the protein by attachment of ubiquitin molecules
2. degradation of the tagged protein into short peptides (7-8 residues) by the proteasome (eliminates protein function)


1. the ubiquitin tag is recognized by the proteolytic machinery of the cell and the target protein is cleaved into short peptide sequences, effectively eliminating protein function

What are the three enzymes involved in ubiquinylation?

* addition of ubiquitinylation to a protein targets that protein for degradation by the proteasome
* 3 enzyme system:


1. E1: ubiquitin-activating enzyme (recognizes ubiquitin in the cytosol and picks it up)
2. E2: ubiquitin-conjugating enzyme (facilitates attachment of the ubiquitin to the target protein)
3. E3: ubiquitin ligase = large family of proteins, each member recognizes a different signal → recognize specific target for degradation and attach ubiquitin to it

What are the steps of ubiquitinylation?

1. ubiquitin activated by linkage to E1
2. activated ubiquitin is transferred to Cys on E2
3. E3 recognizes substrate and transfers ubiquitin to lysine side chain of target substrate
4. poly-ubiquitinylation → adding multiple ubiquitins

What is the structure of the proteasome complex?

* the core forms a central hollow cylinder with proteolytic activity
* caps on each end form narrow openings through which unfolded polypeptides are threatened
* breaks down any protein in the case
* the central cylinder contains the proteolytic enzymes that will breakdown any protein found within this core.

How are proteins degraded by the proteasome?

* the polyubiquitinated protein is unfolded as it is threatened into the proteasome
* the polypeptide is cleaved into small peptides (2-24 aa) which are released and further degraded
* in the spino-cerebellar ataxia, a mutation in the Ataxin 1 gene creates a misfolded Ataxin protein → still tagged with ubiquitin, but does not break down properly and builds up into deadly aggregates

What is a ligand?

* a molecule that is bound by a protein (aka substrate)
* the functions of all proteins depends on their ability to bind with other molecules → if a protein doesn’t bind with anything, it cannot be used
* examples of protein binding include: antibodies binding to target molecules (antigens); enzymes binding to a substrate; DNA‐binding proteins associating with DNA; or receptors on the surface of a cell binding to signaling molecules, such as the growth hormone receptor

What is required for ligand-binding?

* high affinity → strength of binding between protein and ligand
* weak = come together and fall apart quickly
* strong = associated for a long time
* specificity → ability of a protein to preferentially bind to one or a small number of molecules
* both are dependent upon the molecular complementarity between the ligand and the surface of the ligand binding site

What is molecular complementarity?

* dependent upon non-covalent interactions between facing surfaces
* The interactions between proteins A and B and A and C are weak because the shapes of the facing surfaces are poor matches.
* Their shapes prevent the molecules from getting close together and there are few noncovalent interactions (red lines) between the facing surfaces.
* Thermal motion rapidly breaks the molecules apart. The surfaces of A and D are complementary, allowing them to fit closely together, like a lock and key.
* Many noncovalent interactions (red lines) occur between the facing surfaces.
* Though individually weak, the accumulated effect of these interactions allows the molecules to stay together longer despite the forces of thermal motion.
* Both complementarity of shape and the molecular complementarity at these surfaces that allow the formation of noncovalent interactions are important.

Why are protein shape and sequence important?

* Affinity and specificity of protein binding are determined by molecular complementarity.
* On the left, a stable complex is formed when complementary surfaces can get close together to allow a large number of hydrogen bonds, Van der Waals interactions, and ionic bonds to form.
* Note that shape is important, but that the specific amino acids at certain positions are important for molecular complementarity as well.
* The lack of complementarity of shape means that surfaces cannot get close together to form interactions.
* Note also that where surfaces are close together, the lack of molecular complementarity means that non-covalent bonds cannot form, for example where two negatively charged amino acid residues are facing one another

What is a ligand-binding pocket?

* cAMP is an important regulatory molecule that can modulate protein function.
* Proteins controlled in this way have cAMP binding domains.


* In the folded conformation, a binding pocket has formed within the three‐ dimensional structure of the protein.
* In addition, six amino acid residues (coloured) are positioned such the side chains are facing the surface of that pocket.
* Even though these amino acid residues are far apart from one another in the primary sequence, they are all clustering together to form the binding pocket in the folded protein.
* These six amino acids are interacting directly with cAMP to form hydrogen bonds and ionic bonds that hold the cAMP in the binding pocket.
* Once again, the shape of the pocket is important, cyclic AMP fits precisely in this pocket, while ATP, ADP, or cyclic GMP would not.
* Some specific amino acid residues are required to increase the affinity of binding, but not all amino acid residues are directly interacting with the ligand.
* Changing a single amino acid residue may decrease binding affinity by either changing protein shape or eliminating a noncovalent interaction

How is binding affinity measured?

* the free energy of interaction between a protein (P) and its ligand (L) can vary greatly
* binding affinity is measured by the association constant of the binding equilibrium (Keq)
* In the yellow box we have a very simple formula that represents the reversible association between a protein and its ligand.
* Ligand (L) and Protein (P) are on the left
* the Ligand‐Protein complex (LP) is on the right.
* The molecules will cycle between these different states in the solution.
* A high K equilibrium would mean that the reaction would tend to the right where protein is bound to the ligand.
* This would indicate a high‐affinity interaction in which the protein and ligand tend to stay together.
* A low K equilibrium, or a high dissociation constant (Kd), would mean that the reaction would tend to the left.
* This would indicate a low‐affinity interaction in which the protein and ligand readily fall apart

What are Keq and Kd?

* K equilibrium can be represented in the following way: the concentration of the complex divided by the concentration of each of the substrates.
* Dissociation constant is a more common way of representing this reaction.
* K dissociation would simply be the concentration of ligand times concentration of protein divided by the concentration of the complex.
* If we have a low Kd, we would have a high affinity interaction

What is the impact of enzymes on the cell?

* enzymes bind their ligands (called substrates) and promote a chemical reaction between them = catalysis
* Enzymes are proteins that catalyze molecular reactions.
* In the graph, the X‐axis shows the progress of the reaction, while the Y‐axis shows the free energy at different points in the reaction.
* Specifically, we are interested in the change in free energy.
* Lower free energy is favoured, as it is more stable.
* The transition from the reactants on the left to products on the right is favourable since there is a decrease in free energy.
* There is a transition state that has higher free energy.
* In the uncatalyzed reaction, the reactants have to get to this transition state before the products can be produced.
* This is unlikely to happen or will happen at a very low rate.
* The enzyme is reducing the free energy of the transition state.
* Now the reactants can be converted into products with a lower energy requirement.
* The enzyme has not changed the chemistry of the reaction, only the rate.
* What we will consider is how the affinity of an enzyme for the substrates can facilitate the transition state and speed up the reaction

How do catalysis speed up a reaction?

* enzymes can speed up reactions by 10^6 - 10^12 times
* Enzymes increase the rate of a reaction by lowering the energy of activation
* In the uncatalyzed reaction, the reaction can occur, but it occurs at a very slow rate.
* The catalyzed reaction introduces a protein that can specifically recognize both the proteins.
* When the enzyme recognizes those two substrates, it brings them close together facilitating the reaction.
* This is significant when we’re thinking about the lifespan of a cell. If a reaction occurs too slowly, it might be lethal to the cell

What are the two functional regions of the enzyme active site?

1. binding site/pocket - determines the specificity


1. The substrate‐binding site is a part of the active site of the enzyme that also includes the catalytic domain
2. catalytic site - promotes reaction

\*if the structure is disrupted, so is its function

* Enzymes have to demonstrate high specificity and high affinity for their substrates.
* Enzymes have substrate binding sites that demonstrate molecular complementarity for unique substrates.
* Enzymes may act by creating an environment in which the transition state is stabilized, such as bringing substrates close together, or temporarily creating an intermediate state

What is Vmax?

* the catalytic activity of an enzyme is described by Vmax and Km
* Vmax is the maximal velocity of a reaction at saturating substrate concentrations
* the point where the enzymes are fully saturated
* more product can be made as more substrate is added
* We can measure enzyme kinetics in a very simple way.
* One substrate has a low-affinity interaction with the enzyme, the other substrate has a high-affinity interaction.
* The X‐axis represents the concentration of the substrate.
* The concentration of the enzyme is held constant.
* The Y‐axis represents the rate of the reaction, such as the rate at which the product is made.
* With increasing substrate concentration, there is an increase in the rate at which products can be produced.
* At a fixed enzyme concentration, more products can be made as more substrate is added.
* The maximal velocity of the reaction will be achieved at a substrate concentration when all substrate binding pockets on all of the enzymes are, essentially, filled.
* If you compare a low-affinity substrate and a high-affinity substrate with the same enzyme, they will have the same Vmax.
* A fixed amount of enzyme will reach the same Vmax no matter which substrate, as this is the point at which the enzyme’s substrate binding pockets are saturated and the enzyme is working at maximum capacity.
* It will, however, take a higher concentration of low-affinity substrate to get to Vmax since the substrate and the enzyme are more frequently dissociating
* Vmax is unchangeable

What is Km?

* Km (Michaelis Constant) → concentration of substrate at which reaction velocity is half maximal
* Km is a measure of the affinity of an enzyme for the substrate
* Ex. If the Vmax of this interaction is 1, half Vmax is 0.5. If we look at the pink line for the high-affinity substrate we can determine the concentration of high-affinity substrate required to reach half maximum velocity. And this value, known as the Michaelis constant or Km, is the measure of the affinity of the enzyme for that substrate. If we look at the blue line for the low-affinity substrate, we can determine the concentration of low-affinity substrate required to reach half maximum velocity.
* The Km for the lower affinity substrate is a higher value than the Km for the higher affinity substrate.
* Km shows a reciprocal relationship with an affinity

What happens at different enzyme concentrations?

* Changing the concentration of the enzyme has changed the Vmax.
* With one-quarter as many enzymes, there are one-quarter as many substrate‐binding sites.
* The Vmax is limited by the number of available substrate‐binding sites.
* Separate calculations of Km for the two reactions produce the same Km.
* The amount of enzyme present has not changed the affinity of the substrate-binding site.

How does structure dictate function?

* target peptide sequence = arginine - arginine - X (any amino acid) - serine - Y (any hydrophobic amino acid)
* Shown here is Protein Kinase A or PKA. A kinase adds a phosphate group to a target protein.
* PKA has two substrates: the target protein and the nucleotide ATP.
* Represented in this space‐filling diagram, in grey and blue is PKA.
* There is a small domain at the top which includes the glycine lid (highlighted in blue) and the large domain at the bottom.
* A hinge region connects the two domains.
* The two domains together form a nucleotide‐binding pocket (for ATP in green) and a substrate binding pocket (for the target peptide in red).
* These binding domains can be called the kinase core – the site of the catalytic function of the enzyme.
* Molecular complementarity regulates the activity of this enzyme.
* The structure of the ATP‐binding site is specific for ATP and other nucleotides such as ADP, GTP, cAMP bind with a low affinity, readily falling out of the binding site if the drift in.
* The target peptide also binds with specificity to the enzyme.
* The target peptide is recognized by glutamic acid residues found in the large domain.

What are the conformational changes in PKA?

* ATP and the target peptide bind to PKA when the enzyme is in an open conformation.
* Once binding occurs, the small domain and large domain move together due to a change in the shape of the hinge.
* The glycine lid traps the two substrates in place inside the enzyme.
* This also brings the substrates close together, allowing the transfer of phosphate from ATP over to the target peptide.
* Both the target peptide, now phosphorylated, and the nucleotide, now ADP, have new shapes.
* The phosphorylated peptide and the ADP have much lower affinities for the binding sites of PKA.
* As PKA switches back to the open conformation, both molecules readily leave these binding sites.

What are the general mechanisms for regulating protein function?

* allosteric regulation
* covalent modification
* proteolytic cleavage
* signal-induced regulation of protein levels
* compartmentalization
* enzyme complexes

What are allosteric modulators?

* small molecules that bind to sites of a protein to modify function
* exert a positive or negative effect on protein function
* positive modulators (aka allosteric activators) increase activity
* negative modulators (aka allosteric inhibitors) reduce activity

What is the allosteric activation process of PKA?

* the allosteric activator (cAMP) binds to regulatory subunits (R), produces conformational change, and releases the active catalytic subunits (c)
* Allosteric enzymes have multiple subunits, which in turn means that they have multiple active sites.
* The active and inactive conformations of the enzyme differ in their tertiary or quaternary structure.
* PKA switches between two conformations: an active monomer and an inactive tetramer. Inactive PKA contains two regulatory subunits (R) in green and two catalytic subunits (C) in yellow.
* Tetrameric PKA is inactive because the substrate‐binding site (catalytic site) is blocked.
* A domain on the regulatory subunit called the pseudo‐substrate, has a similar shape as the normal substrate.
* The pseudosubstrate binds in the catalytic site, preventing substrate binding.
* This effectively inactivates the enzyme. The small molecule cAMP acts as a positive modulator or allosteric activator.
* cAMP can bind to the regulatory subunits at the nucleotide-binding sites.
* A conformational change occurs to the regulatory subunit upon cAMP binding.
* The pseudo-substrate changes shape so that it no longer binds to the catalytic site.
* This releases the catalytic subunits from the tetramer and renders them active.
* The concentration of cAMP regulates the activity of the enzyme through these regulatory subunits.
* At low concentrations of cAMP, PKA is inactive and at high concentrations, PKA is active

How does allosteric inactivation work with aspartate transcarbamylase?

* cytosine triphosphate (CTP) is an allosteric inhibitor
* The enzyme, aspartate transcarbamoylase or ATCase, is regulated by the allosteric inhibitor, CTP.
* On the left-hand side is the ATCase multimer with six catalytic (yellow) and six regulatory (green) subunits.
* The regulatory subunits contain the modulator binding sites for CTP.
* When CTP binds to the regulatory subunits, conformational changes in each twist the entire complex into the inactive conformation, essentially hiding or masking the substrate binding sites.
* When CTP concentrations are low, CTP binding sites are empty and we see a change in the active conformation.
* The active state, relaxed state (R) and inactive state, or tense state (T) are determined by the concentration of the allosteric modulator, CTP.

What is the process of allosteric regulation and negative feedback?

* this reduces waste
* resources are conserved
* an enzyme catalyzes an early step in a multistep pathway and is inhibited by the final product of the pathway
* Allosteric inhibition is a good way of providing Negative Feedback in a metabolic pathway, that is, turning off a metabolic pathway using the end product as a cue.
* Enzyme 1 is allosterically modulated by the end product, E.
* In this way, the cell does not waste resources and energy by proceeding through this whole pathway if it does not need any of product E.
* This is illustrated by the enzyme aspartate transcarbamylase (ATCase).
* The ATCase enzyme is used in the second step in this pathway. CTP is a negative modulator of this enzyme. As a result, if the concentration of CTP is high in the cell, the cell does not need to make any more. It is the presence of CTP itself that prevents the production of too much CTP.
* This also prevents the unnecessary expense of glutamine and ATP in the cell.

How does allosteric inhibit and activate PKA?

* CTP; allosteric inhibitor, increased Km (reduced affinity)
* ATP; allosteric activator, decreased Km (increased affinity)
* Allosteric enzymes may have many allosteric modulators, both inhibitors and activators.
* While CTP is an allosteric inhibitor of ATCase, ATP is an allosteric activator.
* This graph illustrates the effect that CTP and ATP have on ATCase kinetics.
* Along the X‐axis is the concentration of the substrate, aspartate. Along the Y‐axis is the rate of the reaction.
* The middle line represents ATCase unmodified.
* At point B is the Km of the unmodified ATCase, indicating the affinity between ATCase and its substrate, aspartate.
* The top line represents ATCase with the associated allosteric activator, ATP, The kinetics of the reaction have changed.
* ATCase with ATP has a lower Km or a higher affinity for aspartate.
* The bottom line represents ATCase with the associated allosteric inhibitor, CTP.
* The kinetics of the reaction has again changed. ATCase with CTP has a higher Km or a lower affinity for aspartate.
* Note that while the Km has changed for each activation state, the Vmax remains the same.
* The number of catalytic sites has not increased or decreased

What is co-operative allostery?

* binding of one ligand molecule affects the binding of subsequent ligand molecules
* This is the binding of a ligand to one subunit in a multimeric complex that changes the affinity of all of the subunits for that ligand.
* The stylized graphs demonstrate the significance of this mechanism.
* On the left is a typical enzyme kinetics curve for a monomeric enzyme.
* The substrate concentration is on the X‐axis and the rate of reaction on the Y‐axis.
* Vmax can be used to calculate the Km of the substrate‐protein interaction.
* On the right is the enzyme kinetics curve for a multimeric allosteric enzymes that demonstrates cooperative binding.
* The slope of the curve has changed during the reaction causing an “S”‐shaped curve.

What are the kinetics of co-operative allostery?

* changes the value of Km
* limits the rate of reaction
* occurs in all the subunits
* the rate greatly increases

How does Vmax become reached with small changes in the ligand?

* To get from about 10% reaction velocity to nearly 100% requires a large increase in ligand concentration (from blue star to blue sun).
* In contrast, the kinetics of an allosteric enzyme is shown in red.
* To get from about 10% of reaction velocity to nearly 100% requires a very small increase in ligand concentration (from red star to red sun; boxed).
* This allows the cell to exert powerful and dramatic changes on enzyme activity in the cell, rapidly increasing the efficiency of the enzyme

Why is co-operative allostery useful?

* Cooperative allostery is useful for haemoglobin as it allows the protein to pick up and deposit oxygen efficiently.
* This graph is a representation of the reaction kinetics of two globins: hemoglobin (pink) and monomeric myoglobin (blue).
* On the Y‐axis is the fraction of saturation of hemoglobin.
* Protein activity is measured by the amount of bound substrate.
* On the X‐axis is the partial pressure of oxygen (pO2).
* This is a way of measuring oxygen (substrate) concentration.
* We are interested in the difference in protein activity at two concentrations: in the lungs where oxygen is picked up and in the tissues where oxygen is deposited.
* We can compare the amount of saturation at 2 different partial pressures: one that is typical of the lungs (100 torr) and the other typical tissues (20 torr).
* Myoglobin does not show cooperative allostery. A typical kinetics curve is seen.
* Km can be calculated to be about 35 torrs. For myoglobin, the difference in saturation in the two tissues is 38%.
* The protein is not fully saturated in the lungs and so is not efficiently carrying oxygen.
* In contrast, hemoglobin shows a sigmoidal curve typical of allosteric cooperativity.
* In the lungs, the hemoglobin is saturated ‐ the hemoglobin has such a high affinity for oxygen that the binding sites are filled.
* When hemoglobin moves to the tissues, we find that most of the oxygen is released.
* The difference in saturation in the two tissues is 66%.
* Across a relatively small difference in oxygen concentration, hemoglobin’s affinity for oxygen has changed dramatically, allowing it to rapidly pick up the substrate.

What are covalent modifciations?

* phosphoregulation → an “on-off switch” for enzymes via the addition or removal of chemical groups
* amino acids that are targeted include serine, threonine and tyrosine
* Covalent modification comes in many different forms: acetylation, methylation, carboxylation, etc.
* Phosphorylation is a common modification for all proteins.
* Phosphoregulation is the term used for the reversible addition and removal of a phosphate group on a protein that activates and inactivates protein function.
* One or multiple amino acid residues may be phosphorylated.

What is phosphorylation?

* phosphorylation results in the addition of 2 negative charges
* In the conformation on the left, the inactive protein, CDK, cannot bind to the substrate because the substrate‐binding pocket is blocked by the red domain.
* After phosphorylation, the red domain moves, opening up the substrate‐binding pocket and activating the protein.
* An increase in negative charges with phosphorylation allows the red domain to form new ionic interactions and change protein shape.
* In this case, phosphorylation is an activating event.
* Every type of protein in all cells has the potential to be regulated by phosphorylation.