NOTE: THIS HANDOUT IS NON-EXCLUSIVE AND MAY CONTAIN SOME INFORMATION THAT IS NOT REQUIRED FOR YOU TO KNOW, AND IT MAY BE MISSING INFORMATION THAT IS REQUIRED. UNFORTUNATLY, I CAN NOT PREDICT OR DETERMINE EXACTLY WHAT IS GOING TO BE ON THE EXAM SO THIS HANDOUT IS JUST SUPPLMENTARY. THANK YOU

Week 7

Enzyme: increases rate of chemical reaction, decreases activation energy

• How? Binding to the transition state of the substrate (L. Pauling 1946)

Chemical reactions -> proceed only in the direction of a loss of free energy.

E.g. Glucose when combined with oxygen will spontaneously burn into H20 and CO2. But why does the sugar in the cabinet catch on fire. Because the activation energy barrier is too high and energy needs to be added to the substrates before they can get over the barrier.

When stuff burns -> the molecules are dispersed which increases the entropy of the molecule which is energetically favored and is a loss of free energy. I.e. the products free energy state is lower than the ground state of the substrate.

Enzymes -> Put energy into the substrate to allow them to overcome the activation energy barrier.

- Do this over and over again and very fast and efficient.

- Enzymes are catalyst (substances that lower the ∆G≠ “activation energy”)

- Ground state = the contribution to the free energy of the system by an average molecule (S or P) under a given set of conditions.

- Transition state = fleeting molecular moment. Not the same as an intermediate.

-Catalyst affects the rate in both directions. Just reach equilibrium faster by increasing the rate.

- E+S -> E:S -> E:P -> E+S

-ES and EP are intermediates.

Active site -> pocket that is lined by specific amino acids

Interaction between a.a. + substrates that allows the RXN to go forward. How? -> Selectively stabilize the transition state (fleeting molecular moment) Enzymes have a higher affinity for the transition state than they substrate or product. In the active site they, align various reacting groups, stabilize transient unstable charges and,

rearrange bonds This lowers the activation energy barrier. The energy cost of activation is made up for by favorable interactions during the transition state,

which pays the energy required for the activation barrier. This is known as the binding energy, which is released during the Enzyme substrate interaction

in the transition state. The binding energy is a combination of forces, the same forces that stabilize protein structure

-> hydrogen bonding, ionic interactions, and hydrophobic interactions.

This binding confers both specificity and catalysis. These weak interactions are optimized during the transition state of the reaction. Enzymes are

not complementary to the substrates but to the transition state substrates pass during conversion to products.

Important: Binding energy is the major source of free energy used by enzymes to lower the activation energy of reactions.

JUST A SIDE NOTEI. Evidence for transition state complementarily.

Transition state analogs ->bind it an enzyme more tightly than substrate.

-Can be used to design pharmacological agents such as enzyme inhibitors.

Catalytic Antibodies -> antibodies that can catalyze and enzymatic reaction

Usually antibodies do not catalyze reactions, only bind things. However catalytic antibodies are produced when animals are immunized with hapten molecules

that are specially designed to elicit antibodies that have binding pockets capable of catalyzing chemical reactions.

Using haptens that are transition state analogs -> the antibodies that are created bind an epitope which is the transition state of a chemical reaction. Therefore they are able to catalyze enzymatic reactions.

ENZYMES CONT…

I. ____________(are hydrolases) -> A class of enzymes that use _______ to break ___________ to create two smaller peptides. These include a ______ in the active site.

II. 4 essential and conserved features of serine proteases: 1.) _________ -> This includes 3 key residues –

a. The three residues are very close to one another in the active site, but actually far apart in the 1° sequence.

a) ___ -> Most important in the reaction. Acts as the nucleophile and forms a temporary covalent bond with the substrate.

b) ___ - Acts as a strong base first and then later as an acid. Removes the proton from Ser, turning it into a strong nucleophile.

c) ___ – Helps stabilize the positive charge on His by being a temporary proton acceptor, and turns His into a strong base.

2.) ________ -> 2 hydrogen bonds form that come from main chain N-H form with the negative oxygen on the tetrahedral intermediate. Helps stabilize the transition state.

3.) ___________-> Hydrogen binding a long the main chain of the substrate -> helps to stabilize the transition state.

4.) _____________ - > Complementary to whatever side chains the enzyme cleaves after.

Note: These general features are conserved among many species.

_________ (bacterial protein) – protein with completely different structure, different a.a. sequence, but the active site has the some configuration, and same mechanism, an example of ______________evolution.

• 4 a helices surrounding 5 parallel b-strands -> Alpha/Beta Barrel.

• Active site: C-end of the central b-strands

– Catalytic triad: S,H, D

III. Different types of serine proteases _______ _ -> Cleave after larger positively charged side chains. (K and R) -> Specificity pocket

includes – Asp (small negatively charged) _________ -> Cleaves after small uncharged side chains. (G, A, V) -> Specificity pocket includes

large hydrophobic residues (Thr + Val) ________ -> Cleaves after large aromatic side chains. (F, W, Y) –Specificity pocket includes

Hydrophobic glycine and also a serine.

More detail on chymotrypsin.

Chymotrypsin – cleaves on the C-Terminal after aromatic residues. General mechanism can be broken down into two phases.

- __________ -> In this phase the peptide bond is ________ , and an ester linkage is formed between the substrate and the enzyme. Known as an covalent _________

- ___________-> In this phase _______ enters and the ester linkage is hydrolyzed and the non-acyl enzyme is regenerated.

-Important residues to remember -> Asp 102, His 57, Ser 195. ( don’t think you need to know numbers, not 100% sure.)

Structure of chymotrpysin - > Two domain protein -> Right at the interface where the two domains meet is where the active site is located in the 3-D structure. All the key residues are very far apart in the 1° but come together very close in the active site.

• 2 domains

• Each domain: antiparalled b -barrel, six b -strands

• Active Site: 2 loop regions from each domain

IV. CHYMOTRYPSIN Mechanism -> FOR FIGURE SEE ATTACHMENT AT THE BOTTOM

NOTE: This mechanism and the figure attached at the bottom is a little more detailed than the book. However I feel it’s a little more helpful to try to go through the full mechanism a couple times. You can try it once or twice; you don’t have to memorize everything but DO know the level of detail the book presents.

1. Upon binding of the target protein, the carboxylic group (-COOH) on Asp102 forms a low-barrier hydrogen bond with His57, increasing the pKa of its imidazole nitrogen from 7 to about 12. This allows His57 to act as a powerful general base, and deprotonate Ser195.

2. The deprotonated Ser195 serves as a nucleophile, attacking the carbonyl carbon on the C-terminal side of the residue and forcing the carbonyl oxygen to accept an electron, and transforming the sp2 carbon into a tetrahedral intermediate. This intermediate is stabilized by an oxanion hole, which also involves Ser195.

3. Collapse of this intermediate back to a carbonyl causes His57 to donate its proton to the nitrogen attached to the alpha carbon of the leaving peptide. The nitrogen and the attached peptide fragment (c-terminal to the F W or Y residue) leave by diffusion.

4. A water molecule then donates a proton to His57 and the remaining OH- attacks the carbonyl carbon, forming another tetrahedral intermediate. The OH is a poorer leaving group than the C-terminal fragment, so, when the tetrahedral intermediate collapses again, Ser195 leaves and regains a proton from His57.

5. The cleaved peptide, now with a carboxyl end, leaves by diffusion.

V. Some ways to study enzyme mechanisms:

___________ -> Change or delete a ___________ and see how it effects the enzyme or mechanism

Eg. Chymotrypsin -> eliminate serine, reduce catalytic activity by 2 million times.

- Eliminate his/asp – reduce catalytic activity by 300,000 times. - Asp only – 30,000 times.- Discover the ser is the most important.

VI. Enzyme Regulation:

1.) Allosteric control (other space) _____________ : Mechanism where enzyme activity is__________ either increase (+) or decreased (-) by reversible non-covalent binding of some molecule at another site distinct from the active site. Usually multi-subunit and has a regulatory_______ subunit and _________catalytic subunit.

-The Allosteric modulator or effector can be a small metabolite or co-factor. -This binding causes _______ a Conf. change in the active site of the enzyme

-This type of regulator is often found in metabolic pathways -> feedback inhibition.

THE GREY STUFF IS NOT IMPORTANT TO KNOW I DON’T THINK

2.) Reversible covalent modification__________: Many types (ADP-Ribosylation, Methylation,etc.), vast majority is ____________phosphorylation.

->Kinases ________: Use ATP to add a terminal phosphate onto a Tyr, Ser, Thr or rarely His.

- Can be __________active or non-active when phosphorylated. - Many enzymes are regulated by multiple phosphorylation -> fine tuning________ of control.

(not always binary on/off).- Can be ________hierarchical.- Kinases recognize___________ consensus sequences -> e.g. PKA – X-R-(R/K)-X-(S/T)-B-

->Phosphatase_________: remove phosphates. (less specific)

IMMUNE SYSTEM

• Immune system recognizes, destroys and remembers foreign parasites while ignoring the host’s cells and proteins

• Recognizes foreign molecules = Antigen

– B cells release Immunoglobulins to circulation

– T cells have T cell Receptor that recognizes when presented on MHC class 1 protein

IMMUNE SYSTEM

Important functions -> Distinguish between self and non-self

1.) Specific recognition of foreign particles

2.) Ability to destroy foreign microbes3.) Memory -> allow more of a rapid + stronger response the 2nd time.

Two main branches

1.) _________ Humoral (fluid) -> bacteria and viruses in blood (antigens) + antibodies2.) ________ Cellular - > cells infected with viruses, T-cell receptors interact with MHC molecules that present

viral peptides to the T-Cell.

Definitions:

_______ - any molecule of pathogen that can elicit an immune response.

_____________ specific site on antigen that specifically recognized by the immune molecule. (B cell receptor)

________ ___(fill) -> proteins present in the bodily fluids of vertebrates used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.

How does body make anybodies?

Structure of a classic antibody -> The Immunoglobulin G (IgG).

Structural features-> List all structural features of an antibody.

Specifity of an IG determined by

1.)

2 )

Induced Fit -> enzymes are flexible -> antigen binding causes the shape of the antibody to change inducing a better fit.

Cell mediated response -> Cell infected by a virus presents viral peptides onto its MHC protein -> The T Cell receptor on the T-Cell reads those peptides.

MHC = M_____ H_____ C______ -> found on virtually all our cells. (polymorphic)

Two main MHC classes.

Class I MHC proteins mainly present foreign peptides to cytotoxic T cells

class II MHC proteins mainly present foreign peptides to helper and regulatory T cells

MHC Class I -> Structure = 2 poly peptide chains -> 4 extracellular domains.

Heavy chain -> 3 domains – α1, α2, α3

Light chain -> 1 domain -> Beta2

- Each of the two domains closest to the plasma membrane (a3 and b2)resembles a typical Ig domain while the twodomains farthest from the membrane (a1 and a2) are very similar toeach other (alpha beta domains) and together form a peptide-binding groove at the top of

• All TCRs recognize a helices of MHC • Foreign peptide lays in cradle between helices• High specificity binding of TCR to foreign peptide within MHC cradle• Recognition motif is 2 a helices + 4 antiparallel b strands• Recognition motif connected to TM domain by b sandwich • MHC class 1 is monomeric • MHC class 2 is dimeric

MEMBRANE

A hydropathy plot of the amino acid sequence of an erythrocyte membrane protein begins with a region of high negative hydrophathy index, followed by three regions of high positive hydropathy index, and ends with a region of high negative hydropathy index. (Each of the regions of high positive and high negative hydropathy index spans more than 20 residues.) What can you predict about the topology of this membrane protein?

A. It has three transmembrane domains.

B. At least one domain of the protein faces inside the cell.

C. It is a peripheral membrane protein.

D. It has two domains that span the membrane.

I. BIOLOGICAL MEMBRANES a. BASIC PHOSPHOLIPID STRUCTURE

II. a. Made up of proteins and lipids -> protein/lipid composition can vary among cell types.b. Membranes are very ______, around _____ nanometers, or _____ Angstromsc. Membrane proteins are harder/easier? to crystallize and need to be removed and

crystallized with detergents, which mimic the hydrophobic environment of the lipid bilayer.

d. The lipid bilayer is held together by mostly non-covalent interactions, primarily -> ______________

e. Membrane Lipids are _________ molecules -> have both a polar and a non-polar sidei. Soap and detergents are amphipathic like membranes, eg. Sodium dodecyl

sulfate (SDS). f. 3 Main Types of membrane lipids: 1.) Phospholipids 2.)Glycolipids 3.) Cholesterol

i. 2 types of phospholipids: 1.) Glycerophospholipids 2.) Sphingophospholipids The four main specific phospholipids in membranes :

g. Phosphatidylethanolamine, Phosphatidylserine (-), Phosphatidylcholine, SphingomyelinIII. Detergent vs. phospholipid

The detergent will form a micelle because the single fatty acid tail -> allows for tighter packing

into the core

The phospholipid will form the classic bilayer. A liposome is an artificial lipid bilayer

IV. Cholesterol -> Planar, 4 steroid ring with a polar hydroxyl –OH head group. Ampipathic.a. Abundant -> up to 50% of membrane. (vary among cell types)b. Both increases and decreases the fluidity of membranes -> allows for tighter packing of

the phospholipids, and at the same time prevents crystallization of the phospholipids at low temperatures. -> NOTE ACCORDING TO MONTALS SLIDE HE ONLY MENTIONS THAT CHOLESTEROL MAKES MEMBRANES MORE RIGID.

VII. Asymmetry, Movement: a. Membrane is very asymmetric: eg. PS is only found on the inner leaflet -> important for

cell signaling. (inner is negative). Glycolipids are only found on the outside.b. Movement: 3 types:

1.) uncatalyzed trans bilayer -> rare and slow 2.)Rotation and flextion

3.) uncatalyzed lateral diffusion -> lots and fast , single phospholipid goes all the ways around a cell in 1 sec.

Fluidity : Affected by_________________ and __________________a. ______________________-> temp. where lipids go from fluid to the para-crystaline state. b. Composition of lipids -> degree of saturation of the hydrophobic tail

i. __________________= higher phase transition temperatureii. _________________ = _____________________-> lower phase trans. Temp

iii. Hydrophobic tail length -> ______________ = higher phase trans. Temp.iv. Why -> with more saturation, lipids can pack more tightly. Double bond introduces

kinks.v. ________________ can change lipid comp with varying environmental

temperatures. c. Cholesterol -> Modulator, prevents crystallization at temp below phase trans. Increases

rigidness at temp above phase trans.i. How? Inhibit the close packing of the hydrophobic tails.

d. Temperature ->i. Low temperature membranes become less fluid.

ii. High temp membranes become more fluid.

Question: would you expect a polar bear to have more/less unsaturated fatty acids that a cold water fish? Why?

e. Integral Membrane Proteins -> Amphiphatic , transmembrane proteinsi. Firmly associated with the membrane via lots _______________interactions.

ii. Can be removed via ________________iii. Can be ____________ or __________________iv. Allow the cell to communicate with the outside to the inside of the cell.

a. Integral Membrane proteins -> are usually beta barrels or alpha helices. Why?

b. Integral membrane proteins are harder to______________.c. __________________ : Experimentally determine the polarity of amino acid residues. ->

move from a non-polar solvent to a polar one. Determine Free E change.i. ___________ -> hydrophilic

ii. ___________ -> hydrophobic d. __________________ -> used to predict regions that might _____________________

i. Plot hydrophathy index ( y axis) vs. Residue number (x-axis). ii. Average length of transmembrane alpha helix = __________ residues.

Can extract/ isolate integral membrane proteins and study in artificial membranes

BACTERIORHODOPSIN

Found in the membrane of bacterium Halobacterium Halobium -> 248 a.a. and binds retinal, the same photosynthetic pigment we use in our eyes to capture light.

Bacteriorhodopsin uses light energy to pump protons from the cytosol to the extracellular space.

7-Transmembrane helices span the hydrophobic membrane -> The helices are tilted about 20° in respect to the membrane -> characteristic of a helical bundle.

When retinal absorbes a photon it undergoes a trans to cis isomerization at bond position 11 and protons are subsequently pumped across the membrane.

Retinal is bound in a pocket of bacteriorhodopsin, and the pigment forms a Schiff base with a lysine residue -> LYS 216. i.e. it is covalently linked to the nitrogen atom of a lysine side chain that is protonated and therefore has a positive charge.

The lysine is positioned in a channel near the cytosolic side that is lined with hydrophobic residues with the expectation of a asp 96 which is important for pumping protons -> the extracellular side of the channel is wider and is lined with hydrophilic amino acids including asp 85

Tense (T) – binds trans -retinal Relaxed (R) – binds cis- retinal

PROTON RELAY MECHANISM

Short version

R state: H+ transferred from Asp 96 to Schiff base and H+ from Asp 85 is transferred to Extracellular Space à H+ transferred from Cytoplasm to Asp 96

Long Version

Tense State binds trans-retinal -> photon hits and retinal is coverted into cis, this causes to the Schiff base to change its position relative to the Asp85, whicn induces transfer of the Schiff base proton to the Asp86-> once this transfer has occurred to protein undergoes a T State to R state conformational change -> Asp 85 then delievers the extra proton to the EC space, and Asp 96 reprotonates the Schiff base, which subsequently reverts to the trans state and the protein goes back to the T state ready for another cycle.

Why is retinol purple in the membrane but orange when outside the membrane?o Because the environment is different when its in the membrane.

PORIN Protein BETA BARREL

Generally found in bacteria Homotrimeric protein. Each monomer is made of a 16 stranded Up/Down β Barrel. Subunits interact by polar loop and hydrophobic side chain interactions. Periplasmic End: short and smooth Cytoplasmic End: funnel shaped, made of long loops with hydrophilic residues. Eyelet = loop connecting β5 and β6 extending into the central open cavity of the barrel. Cavity is lined with charged residues, arranged in an electrochemical gradient. Different from RBP, because this time the inner barrel is hydrophilic to allow polar molecules to

pass through the membrane, and the outside is hydrophobic to interact with the hydrophobic

membrane. Notice the alternating hydrophobic hydrophillic residues, similar to RBP but just reversed.

Aromatic Belt – mainly _________and_________

has polar and hydrophobic character to interactive with the hydrophilic space of the membrane extracellular solution and hydrophobic internal space of the membrane.

Helps stabilize protein in membrane.

Bacterial. K+ potassium channel (KCSA)

i. 4 identical transmembrane units, which form a central pore -> homotetrameric ii. Functional Channel requires four subunits.

iii. Each monomer is made of 2 transmembrane α-helices, 1 pore helix and a cytosolic tail.

iv. Selectivity Filter – formed from the backbone carbonyl Oxygens from the loops connecting pore helix to the inner transmembrane helix. The structure is stabilized by the packing of residues from the loop into the pore helix.

v. _________________ residues concentrate on the cytsolic side to attact potassium and repel __________ Selectivity filter ___________ carbonyls are precisely aligned so they ____________ favorable interaction with dehydrated potassium. Sodium is too ___________, carbonyl interactions not favorable enough.

K (OH2)8+ = hydration shell (8 H2O needed to stabilize K+ ion)o Within the K+ Ion Channel only:

Resolvation Energy ≥ Desolvation Energy Na (OH2)6+ = hydration shell (6 H2O needed to stabilize Na+ ion)

o Within the K+ Ion Channel only:Resolvation Energy <<< Desolvation Energy

As a K+ ion enters the selectivity filter, repulsive force from the next K+ ion entering the filter will push the first ion through the protein out of the cell.

Some distances: selectivity filter is 3A wide, and 12A long. The protein is about 34A across the membrane, and the bigger opening is 10A wide.

K channel Images.