Biochem Unit 2 Exam

What is the primary difference between enzymes and antibodies?

Antibodies bind a stable chemical structure

What are enzymes?

Proteins that catalyze reactions

• Lower activation energy, but do not alter the energetics of reactions

• Stabilize the reaction intermediate

• Optimized to act selectively on one specific substrate

• Typically produce a single product

• Often regulated

How do enzyme increase reaction rates?

1. Orienting substrates

2. Promoting substrate proximity

3. Constraining structural vibrations

4. Stabilizing intermediates

What does ΔG^Ŧ predict?

How fast equilibrium is reached

What does K_m predict?

Time dependent equilibrium for [ES]

What are the 5 Michaelis-Menten Model Assumptions?

1. Simple Binding Mechanism

2. Initial rate conditions [P]≈0, negligible reverse reaction

3. Steady State: [ES] is constant

4. Free Ligand: ([S] » [E], so [S] ≈ [S] + [ES])

5. Enzyme conserved

If substrate concentration is doubled, does V₀ also double?

No, it will go up but not double

If graph is Time vs Product concentration?

V₀

If graph is Substrate concentration vs Initial velocity?

K_m

If graph is Substrate concentration vs ES complex?

K_d

All the different K’s table

What is Kcat?

K_cat is the turnover number of an enzyme, often on ‘rate limiting’ step

What is the best way to compare catalytic efficiencies?

Look at the ratio K_cat / K_m.

• This is termed the specificity constant or enzyme efficiency

• high Kcat good

In V_max = K_cat[E_total], what is rate limiting

K_cat

What is Line-weaver Burke equation?

What are the 3.5 kinds of enzyme inhibition?

• Competitive

• Uncompetitive

• Mixed

  • Special mixed - Non competitive

Inhibition trends table

What are the 4 structural contributions to rate enhancement

1. Promoting substrate proximity (2-5 fold enhancement)

2. Orientation of substrates (2-5 fold enhancement)

3. Constrict structural vibrations (up to 10⁸ fold enhancement)

4. Stabilizing intermediates (up to 10⁸ fold enhancement)

What are the catalytic triad and mechanisms like between different Serine Proteases?

All Serine Proteases have catalytic triad and nearly identical mechanisms

What is Chymotrypsin

Type of Serine protease

What are the two kinetic steps of Chymotrypsin? (Also draw it)

1. Acylation

2. Deacylation

What is the catalytic triad of Chymotrypsin? What does it do?

Serine, Histidine, and Aspartate in close proximity to each other

• Prepares nucleophile

What is the hydrophobic/substrate recognition pocket of Chymotrypsin? What does it do?

What binds the bulky/aromatic group

• Provides specificity

What is the oxyanion hole of Chymotrypsin? What does it do?

Stabilizing the intermediates, since the intermediates have the negatively charged oxygen that is bound in the oxyanion hole.

What are the 7 steps for Chymotrypsin cleavage?

1. Substrate binding (hydrophobic pocket)

2. Nucleophilic attack by Ser

   • Made possible because Ser is deprotonated due to close proximity of His (acts as a general base), and protonated/positively charged His is stabilized by Asp

   • Oxyanion hole stabilizes after nucleophilic attack

3. Release of first product/ half the peptide that is not bound by the hydrophobic pocket (C=O double bound reformed as well)

4. Water enters active site and is activated by His and Asp

5. Water attacks carbonyl carbon (Hydrolysis of acyl enzyme)

   • oxyanion hole stabilizes intermediate again

6. Bond by Ser and peptide breaks, and C=O double bond reforms (Reforming catalytic Serine)

7. Release of second product by hydrophobic pocket

What are lipids function?

• Fuel and enrgy storage

• Insulation/water proofing

• Membrane components

How many double bonds does a saturated lipid have

0 double bonds

If you want a more rigid membrane, what kind of lipid will you want

Saturated

If you want a more fluid membrane, what kind of lipid will you want

Unsaturated

is the melting point lower or higher for saturated lipids

Saturated lipids have a higher melting point than desaturated lipids

Draw the structure of a Diacylglycerol

Diacylglycerols promote what?

Diacylglycerols promote membranes

C-3 is what for diacylglycerols?

Head group

Why can steroid hormone receptors function inside the nucleus rather than on the plasma membrane?

Steroid hormones are hydrophobic, thus can cross the membranes

How is the lipid desaturation percentage for organisms in hot springs different from human lipids?

Decreased desaturation, because it would allow for increased surface area alignment, decreases mobility, and creates a more stable, solid membrane structure at high temps

What membrane behavior is changed by desaturation?

Lateral Fluidity

What do flippases do?

Move outer phospholipid to inside of membrane

What do floppases do?

Move inner phospholipid to outside of membrane

What does scramblase do?

Move lipids in either direction, toward equilibrium

Tracking of single-particle tagged lipids show “hopping” between regions. What could form these “molecular fences”?

• Membrane regions of different lipid composition (“lipid rafts”)

• Large protein-protein complexes in the membrane

• Intracellularly-anchored membrane proteins

What is the driving force behind membrane lipid rafts and lateral movements?

Brownian motion

What are the 3 molecules and kinds of passive transport?

All go down an electrochemical gradient

• O_{2 -} passive diffusion

• Na⁺ - Diffusion through an aqueous channel

• Glucose - Facilitated diffusion with a carrier protein

What are the two components to electrochemical gradients?

• Concentration (moves from high concentration to low concentration)

• Charge

In this equation, what is C₁ and C₂ ? What does C₂/C₁ equal? What is Z, F and ψ?

• C₁ - Where it originates

• C₂ - where moving towards

• C₂/C₁ = Q

• Z - Charge of ion

• F - Faraday’s constant

• ψ - Electrical potential (Volts, V)

How does active transport work?

Directly couples ATP to move solutes against an electrochemical gradient

What are the types of Primary Active Transport?

• V-, P-, and F-type ATPases

• ABC family transporteres

How could you prevent an ABC family transporter from running backwards?

Promoting certain vibrations using a “Brownian ratchet”

What is Secondary Active (Facilitated Diffusion) transport?

Use ions moving down gradient to facilitate solute moving aginst its gradient

What are the types of Secondary Active (Facilitated Diffusion)?

• SLC family proteins

• Phospholipid scramblases

What are the 3 types of transport systems?

• Uniporter

  • Move 1 thing in either direction, depending on gradient at time

• Symporter

  • Move two things in one direction

• Antiporter

  • Move two things in different directions

What kind of lipids pack tighter?

Saturated Lipids

How is membrane potential produced? What is Resting potential caused by?

• Membrane potential = Sodium-potassium pump (Na⁺/K⁺)

• Resting potential = Various leak channels

How does the Sodium Potassium pump work? What is charge outside and inside the membrane

• Pumping out 3 Na⁺ changes while only letting 2 K⁺ in.

• Keeps inside negative and outside positive

Why is ATP hydrolysis favorable?

• Released phosphate and ADP are resonance stabilized

• Relief of charge repulsion (phosphates are negatively charged)

• Increased entropy

• Cell sequesters ADP to decrease product concentration (LeChatelier’s principle)

• Because has a negative delta G, can compensate for reactions that have positive delta G

What are channelopathies

Diseases caused by mutations in channels

There are different proposed classifications of GPCR’s, with high sequence similarity within a class and low similarity between classes. The differences between GPCR's of the same class is due to what kind of evolution?

Divergent Evolution (Diff function, high homology)

What are the elements of signal transduction

1. Reception

   • Relay

   • Transduce and amplify

2. Transduction

   • Integrate

   • Feedback

3. Response

   • Distribute

What is sensitivity in signal transduction?

The affinity of a receptor for the ligand determines if it can respond to low concentrations of that ligand, or if a higher concentration is needed

What is amplification in signal transduction?

A single signal can trigger a large response by activating many downstream proteins

What is modularity in signal transduction?

• May be a different response from same ligand and receptor depending on pre-existing recruitment

• Signaling pathways have different “modules” (groups of proteins) that can combine in different ways to lead to different responses

What is desensitization/adaption in signal transduction?

A cell’s response can eventually decrease when it begins signaling constantly

What is integration/divergence? What are the two kinds?

1. Integration: multiple signals combine to determine a response.

2. Divergence: one signal branches out to produce multiple responses.

Need two inputs to produce signal

• Logic gates - “And/Or” - need two ligands to make signal, or need just one

• Pleiotropy - one ligand makes multiple types of signal

What is localized response?

• Signal activates but immediately downstream, message remover stops signal. Makes a really fast response

  • Usually happens with scaffolding proteins for all key players in signal

• Signaling produces a response in a specific part of the cell.

Difference between feedback and feedforward loop?

• Feedback - Signal goes off and downstream message comes back and tells signal to turn off

• Feedforward - Signal goes off and downstream message comes back and tells signal to do more

What are the 4 general types of Signal Transducers

1. G protein-coupled receptor

2. Receptor enzyme (tyrosine kinase)

3. Gated ion channels

4. Nuclear receptor

   • hormone binding allows the receptor to regulate the expression of specific genes

What is an agonist?

Molecule (often drug or natural ligand) that binds to receptor and activates it, mimicking the action of a naturally occurring substance

What is an antagonist? What kind of inhibition does it have?

Molecule that binds to a receptor but does not activate it. Instead, it blocks the receptor, preventing agonists from binding and triggering a response

• Competitive inhibition since it has no chemistry

What is a G-protein? What are the two states? What is its intrinsic activity?

• Binds Guanosine nucleotides

  • GDP-bound - inactive

  • GTP-bound - active

• Intrinsic GTPase activity (GTP hydrolysis)

What are GEFs?

GEFs (Guanine nucleotide exchange factor) promote GTP replacing GDP → activity

What are the amino acids that can be phosphorylated?

Ser, Tyr, and Thr

What are the amino acids that can be phosphomimetic?

Asp, Glu

What are GAPs?

GAPs (GTPase-activating protein) terminate activity by promoting hydrolysis

Non-small cell lung cancer is frequently driven by a KRAS G12C mutation that inactivates the GTPase. How does this mutation affect KRAS activity?

GTP cannot be hydrolyzed to GDP, so KRAS is perpetually active

What is the structure of G-protein?

Heterotrimer, a, B, y subunits

• a is homologoous to small GTPase

• Inactive (GDP) bound trimer associates with GPCR

What does Ligand-bound GPCR act as? How does it do it?

Ligand-bound GPCR act as GEF (Guananine nucleotide exchange factor)

• G_a dissociates from GPCR

• G_a and G_by propagate downstream signals

What does RGS (regulator of G protein signaling) act as?

RGS acts as GAPs for G_a

What is name of G_by

Obligate dimer, always bound together

What is the GPCR signaling pathway

•Ligand binds to its GPCR.

•Activated GPCR acts as a GEF, replacing GDP with GTP on Gα.

•Gα–GTP separates from Gβγ and the receptor.

•Gαs–GTP activates adenylyl cyclase.

•Adenylyl cyclase converts ATP → cAMP.

•cAMP binds to regulatory unit of PKA and activates it.

•Active PKA released and phosphorylates downstream proteins.

What is the secondary messenger in the PKA pathway?

cAMP

What is the Phospholipase C (PLC) pathway?

1. GPCR alpha subunit activates phospholipase C (PLC)

2. PLC cleave PIP₂ into IP₃ and DAG

3. DAG stays near the membrane and activates protein kinase C (PKC) which phosphorylates downstream proteins

4. IP₃ goes into the ER and causes it to release calcium, which activates other proteins

What is the secondary messenger in PLC?

IP_3, DAG, Ca²⁺

What are the 5 secondary messengers?

Amplify signal

• cAMP (PKA)

• Ca²⁺ (PLC)

• DAG (PLC)

• IP_{3 (PLC)}

• PIP₃

How does PKA recognize its substrates?

• Has a negatively charged active site

• Phosphorlyate proteins at 3 aa residues (all have OH group, PKA looks for hydroxy to add phosphate to)

  • Serine

  • Tyrosine

  • Threonine

Kinases phosphorylate other proteins at S, T, and Y residues. How could you mutate these residues to create a “perpetually” phosphorylated enzyme?

1. G

2. E

3. R

4. A

E (glutamate) because it can mimic the negative charge of a phosphate group, making the enzyme behave as if it is phosphorylated

Negatively-charged amino acids are sometimes used to “mimic” phosphorylation. Which of the following is a difference between these mimics and authentic phosphorylation?

A. Geometry

B. Charge density

C. Phosphoester bond

D. Can be removed by Phosphatase

E. All of the above

E. All of the above

What are RTKs (Receptor Tyrosine Kinases)?

Extracellular receptors that auto transphosphorylate their own tyrosines in dimers

How does Autophosphorylation work with RTKs

1. Dimeric ligand binds extracellular domain of the receptor

2. Receptor dimerizes, activates kinase activity

3. Receptor undergoes trans-autophosphorylation

4. Intracellular substrates are recruited via pY binding domains (SH2 and PTB)

   1. Signaling complex is formed

   2. Downstream signaling cascade is initiated

What does Grb2 do? What is it comprised of

• Grb2 is a Signaling adapter protein

• Has SH2 and SH3 domains

  • SH2 domains - phosphorylated tyrosine

  • SH3 domains - polyproline helix

How does Downstream Insulin Signaling work (example of RTK)

• Growth signal

  • Recruit Shc domain (scaffolding protein), binds Sos, sos binds Ras (GTPase) that causes cell phosphorylation and results in cell proliferation

• Metabolic signal

  • Recruit IRSs domain (scaffolding protein) - binds p85 and p110 and makes PiP₃

How is RTKs general pathway

1. Subunits of the receptor are separate until bound by ligand, and then they dimerize

2. After dimerized, they transautophorphorylate (add phosphate groups to each other) on tyrosine residues

3. Phosphotyrosine provides a binding site for certain domains of other proteins

   • SH2 domain of GRB2

   • SH3 domain of GRB2 binds polyproline helices of other downstream proteins like SOS

With RTKs, what does PI3 kinase do?

PI3 kinase turns PIP2 → PIP3, which can act on downstream effectors

What is an oncogene? What is an example of one?

Activates gene, make to much of something, make cancer

• PI3K is an oncogene and concerts PIP2 to PIP3, which acts as a membrane localized secondary messenger

What is a tumor suppressor

Normally slows cell growth and prevent cancer

Mutation: deactivates gene - gets rid of “break genes” make more of something, cause cancer

• PTEN is a tumor suppressor and catalyzes the reverse reaction, turning off signaling

How do you perform a Proteomics experiment?

Measure PTMs (Post translational modifications)

• Mass Spec

  • MS1 scans give you intact peptide mass, select peptides for fragmentations

  • MS2 (MS) scans give you peptide sequence and PTM localization

How do ATP hydrolysis and phosphoryl transfer drive the shown reaction?

A. Entropy of hydrolysis contributes heat to the reaction

B. Phosphate used to avoid LeChatlier’s penalty

B. Phosphate used to avoid LeChatlier’s penalty, because “Greater stability of ADP prevents the phosphoryl transfer back reaction and no water produced”

What is Keq?

Concentration of products/ Concentration of reactants

What is Reduction and Oxidation?

• Reduction: Decrease oxygen, increase hydrogens

• Oxidation: Increase oxygen, decrease hydrogens

What type of reaction is a dehydrogenase

Oxidation since it removes hydrogens

What does NAD⁺ (Nicotinamide Adenine Dinucleotide) do?

• Accepts 2e^- and H⁺(hydride ion) to generate NADH

• Nicotinamide ring reactive part of molecule

• NADPH used as reducing power in anabolic rxns

What does FAD (Flavin Adenine Dinucleotide) do?

• Accepts 2e^- and 2H⁺ (as hydride ion + a proton, or two hydrogen atoms) to generate FADH₂

• Isoalloxazine ring reactive part