Exam 3 (Final) Flashcards

Question

What is the difference between thermodynamics and kinetics?

Answer 1

Thermodynamics describes the overall properties, behavior, and equilibrium composition of a system; kinetics describes the rate at which a particular process will occur and the pathway by which it will occur.

Answer 2

* A_out ←→ A_in * G_A = RT ln[A] * ∆G_A = G_A(out) - G_A(in) * ∆G_A =RT ln( [A_in] / [A_out] )

Answer 3

* If the concentration of A is greater on the outside of the membrane than the inside: movement of A from out to in is spontaneous/favorable (∆G_A \< 0) * If the concentration of A is greater on the inside of the membrane than the outside: movement of A from out to in is non-spontaneous/unfavorable (∆G_A \> 0)

Answer 4

free energy of A / chemical potential of A

Answer 5

* movement of charged species results in charge difference across membrane (membrane potential) * Membrane potential - difference in charge across a membrane

Answer 6

∆Ψ = -100mv * inside of cells more negative than outside

Answer 7

* movement of uncharged species * both diffusion and charge favor movement in the same direction * concentration and charge are opposing

Answer 8

* non-mediated and mediated

Answer 9

* diffusion across cell membrane (passive) * high to low concentration * rate of diffusion depends on ∆conc and solubility in membrane

Answer 10

* carrier protein is involved * 2 types 1. Passive/facilitated diffusion: high → low concentration, molecule forms channel 2. Active mediated transport: low → high concentration, coupled to exergonic process (ATP or coupled w high → low)

Answer 11

* ionophores * porins * ion channels * aquaporins * transport proteins

Answer 12

* peptides that increase permeability of membrane to ions 1. Carrier Ionophores - e.g. Valinomycin: K+ is bound by carbonyl groups in valinomycin, then on outside have hydrophobic valine side chains that allow it to pass through 2. Channel forming ionophores - e.g. Gramicidin: K+ ions travel up this channel

Answer 13

* beta- barrel structures with aqueous channels * size of the channel and the residues that line the channel determine what is transported

Answer 14

* Maltoporin: transports maltodextrin (an alpha (1-4) linked glucose * has aromatic groups that bind to the hydrophobic face of maltodextrin on one side * has a greasy slide face on one side of the membrane that allows it to slide through

Answer 15

Na+, K+, Cl-

Answer 16

* KcsA * K+ channel, made of an integral membrane protein * alpha helical structure * right at the entrance to the channel are negatively charged residues that attract K+ * there are neutral residues inside once it enters the channel * has a selectivity filter that lines the inside of the channel

Answer 17

* Selectivity filter is TVGYG sequence that lines the inside of the ion channel * initially K+ is hydrated and when it gets to the S.F. K+ gets dehydrated * carbonyl O from residues lining the channel replaces the hydration shell * selectivity comes from the fact that Na+ is too small for interaction with carbonyl O - can't use this channel

Answer 18

gated - selectively opened and closed

Answer 19

* mechanosensitive - responds to deformities in the lipid bilayer * ligand-gated - respond to extracellular signal e.g. neurotransmitters * signal-gated - intracellular binding to signal * voltage-gated -responds to change in membrane potential

Answer 20

* ligand gated and voltage gated

Answer 21

* Na+ has a higher concentration on the outside of the cell * K+ has a higher concentration inside the cell

Answer 22

1. Resting potential of membrane is normally at -60mv 2. Neurotransmitter release 3. Opens ligand-gated Na+ channels, Na+ travels from out → in 4. Membrane potential becomes positive 5. Voltage-gated Na+ channels open 6. Membrane potential spikes (+20mv): called the action potential 7. Voltage gated K+ channels open after a lag, K+ travels from inside to outside 8. Membrane potential decreases, if it dips below resting potential it is called hyperpolarization

Answer 23

* K_v channel - a type of voltage-gated K+ ion channel * N-term cytoplasmic domain * Transmembrane domain has 6 helices S1-S6 * C-term cytoplasmic domain

Answer 24

* S4 Helix contains 5 positively charged side chains which function as a _voltage sensor_ * When membrane potential increases → S4 Helix moves → S4-S5 linker moves → S5 and S6 move to form pore * K_v channels also have a _second gate_: which ensures that channels close a few minutes after opening * the second gate happens at the N-term inactivation ball → when the channel opens the ball unravels itself and snakes its way into the pore of the channel

Answer 25

* type of passive mediated transporter * transports H2O across membrane * exists in tetramers where there are four channels in one aquaporin * at its narrowest point width = 2.8Å = vanderwaal's radius of water (size selection) * pore is lined with hydrophobic residues inside (charge selectivity) * His+Arg residues disrupt H-bonding between H2O molecules, prevent H+/H3O+ transport

Answer 26

* e.g. GLUT1 glucose transporter * glucose binds, bottom opens and top closes, glucose dissociates and is delivered inside * may be uniporters, symporters or antiporters

Answer 27

transport a single type of molecule at a time e.g. GLUT1

Answer 28

Transport two different molecules in the same direction

Answer 29

transport two different molecules in opposite directions

Answer 30

* endergonic * against concentration gradient * coupled to ATP hydrolysis

Answer 31

1. P-Type ATPase - undergo phosphorylation during transport Na+, K+, Ca2+ 2. F-Type - H+ in mitochondria/bacteria 3. V-Type - H+ in vacuoles/lysosomes 4. A-Type - anions 5. ABC transporter - ions, metabolites, drugs

Answer 32

* Na+-K+ ATPase → move Na+ out and K+ in to cells (opposite of ion channels during nerve impulses)

Answer 33

* Exists in an E1 and E2 state * In the E1 state a glutamate is attached to the protein (facing inside of the cell) * ATP binds, and 3 Na+ bind from inside the cell * ATP is converted to ADP and the E1 is converted to E2 which prefers to face outside of the cell * The Na+ are released outside of the cell, 2 K+ bind the E2 state and phosphate is released * When phosphate is released the enzyme returns to the E1 state which prefers to face inside the cell * The K+ are transferred to inside the cell and the cycle repeats

Answer 34

* a p-glycoprotein - a protein that pumps drugs out of the cell * this is also known as a multi-drug resistance transporter * there are 2 ATP binding pockets * drug binds to transporters and gets kicked back out * CFTR is an example

Answer 35

* resistant cancer cells often over-express p-glycoproteins

Answer 36

* protein-based catalyst * increases the rate of a reaction (impacts kinetics) * catalyst not consumed, always regenerated at the end of reaction cycles

Answer 37

* _high reaction rate_ accelerations: 10⁶-10¹⁴ times the rate of uncatalyzed rxn * _high specificity_ - geometry and charge of active site * can be _stereospecific_ * some enzymes use _cofactors_

Answer 38

* Examples of cofactors include metal ions and prosthetic groups (heme, PLP, biotin) * apo-enzyme (inactive) + cofactor → holoenzyme (active)

Answer 39

When A-B + C → A + B-C the reaction must travel through a high energy, unstable transition state

Answer 40

* free energy (G) vs reaction coordinate * reaction coordinate: path of minimum free energy which reactants use to form products

Answer 41

* if it is a multistep reaction, the overall rate of the reaction is determined by the largest ∆G⁺⁼ (delta G double dagger)

Answer 42

* catalyst lowers (∆G⁺⁼) activation energy * lowers activation energy for a reaction in both directions - forward and reverse reactions are accelerated

Answer 43

* ∆G⁺⁼uncat - ∆G⁺⁼cat = ∆∆G⁺⁼uncat * this is the change in activation energy between catalyzed and uncatalyzed reactions

Answer 44

1. Oxidoreductases - oxidation/reduction 2. Transferases - transfer of a functional group 3. Hydrolases - breaks a bond using water 4. Lyases - group elimination - C=C 5. Isomerases - isomerize - don't add or remove an atom 6. Ligases - bond formation reaction, typically requires energy input

Answer 45

1. acid-base 2. covalent 3. metal ions 4. proximity and orientation 5. preferential binding to transition state

Answer 46

* position acidic/basic groups in perfect orientation for proton transfer * RNAase A → cleaves phosphodiester bonds in RNA * can cleave 1000s of RNA molecules b/c of perfectly placed basic and acidic groups

Answer 47

* transient formation of a covalent bond between enzyme and substrate (covalent enzyme substrate complex) * Activates nucleophile/electrophile * Nucleophile - e^- ser/thr, tyr, histidine, cysteine, lysine * Electrophile - e^- acceptors * Example: cysteine protease → Mpro in SARS-Cov2

Answer 48

* Catalysts: Fe³⁺/Fe²⁺ Cu²⁺ Mn²⁺ Co²⁺ * Structural: Na⁺ K⁺ Ca²⁺ * Both: Zn²⁺ Mg²⁺ * Catalysts: * orient substrates and shield from build up of negative charges * Example: carbonic anhydrase

Answer 49

* bring substrates into close contact (pseudo intramolecular interaction) * bring substrates together in the proper orientation for reaction to occur * reduces translational and rotational motion

Answer 50

* enzyme binds tightly to the transition state to stabilize it, pushes the reaction towards forming the transition state * stabilizing the transition state serves to lower activation barrier * Proline Racemase is an example

Answer 51

* when designing an inhibitor design a molecule that resembles the transition state more than the reactants * enzyme binds to the inhibitor rather than the transition state * Example: Proline racemase - there are inhibitors that the enzyme prefers to bind to

Answer 52

They use multiple different strategies

Answer 53

* destroys bacterial cell wall (MurNac-GlcNac) * 10⁸ enhancement rate * stabilizes half chair and lysozyme cuts * acid-base, then covalent catalysis * inhibited by f-lactone analog (mimics transition state) * residues are Glu and Asp

Answer 54

* they use serine nucleophiles * cleave amide bonds on proteins * examples are digestive enzymes: * _trypsin_ cleaves next to positively charged residues- Lys/Arg * _chymotrypsin_ cleaves bulky aromatic/hydrophobic (Trp, Phe, Tyr) * _elastase_ cleaves small neutral (Ala, Gly, Val) * also active in the blood clotting pathway: “factor” enzymes inactivate zymogen, have to be cleaved by another protease to form active protease → people with Hemophilia lack one of these

Answer 55

* catalytic triad: Asp - His - Ser * Asp and His activate the serine nucleophile * Covalent catalysis and transition state stabilization * have specificity pockets

Answer 56

* Trypsin - negatively charged Asp interacts with positively charged lysine on the substrate (negative residues, positive substrates) * Chymotrypsin - big pocket * Elastase - small pocket (large residues, small substrate)

Answer 57

* a natural protein inhibitor for trypsin * prevents H2O from attacking the transition state * BPTI remains covalently bound to trypsin

Answer 58

* inactive precursors, have distorted active sites * are activated when cleaved by another protease * Examples: * zymogen of trypsin is trypsinogen * zymogen of Factor X_a is Factor X

Answer 59

* If one enzyme in a pathway is tightly regulated it regulates the flux through the whole pathway * if x is highly active the entire pathway is affected (Le Chatlier's principle)

Answer 60

1. control of enzyme amount 2. control of enzyme activity

Answer 61

* as the concentration of an enzyme increases, the rate of the reaction increases * rate of synthesis and degradation of an enzyme directly impacts reaction rate

Answer 62

1. Changing the concentrations of substrates and products influences enzyme activity through le chatliers principle 2. Product inhibition - a product of a reaction has some affinity to an enzyme, when there are high concentrations it remains associated w the enzyme and prevents its functioning 1. Endogenous inhibitors: BPTI 3. Allosteric effectors 4. Covalent modification: zymogens and phosphorylation

Answer 63

inhibition or activation of an enzyme by a small regulatory molecule that interacts at a site (allosteric site) other than the active site

Answer 64

* Aspartate Transcarbamoylase (ATCase) * catalyzes reaction of: * carbamoyl phosphate + aspartate → carbamoyl aspartate + phosphate * this is the first step of pyrimidine synthesis in the body

Answer 65

* C₆R₆ one unit is made up of 6 catalytic and 6 regulatory units * the catalytic subunit alone is active, and has typical enzyme properties * hyperbolic binding curve * when the C₆R₆ unit is formed, the enzyme has unusual properties * sigmoidal binding curve * cooperative enzyme - binding of the substrate to one catalytic unit increases the substrate affinity to other catalytic units within the C₆R₆ complex

Answer 66

* Enzyme exists in T - R * T - inactive * R - active * **_CTP_** * When carbamoyl phosphate is produced it goes through a pathway eventually releasing CTP * High CTP levels lead to feedback inhibition of the first reaction * CTP decreases enzyme activity * **_ATP_** * ATP is a purine, want your rates of purine and pyrimidine synthesis to be coordinated * When ATP levels are high, and CTP levels are low you want to activate ACTase to make more pyrimidines - ATP increases enzyme activity

Answer 67

* ATP and CTP bind _regulatory units_ on C₆R₆ * CTP binds to T state * ATP binds to R state * When ATP binds it changes the contacts between subunits and changes it to the R-state

Answer 68

* phosphorylation * Ser/Thr/Tyr residues are phosphorylated by protein kinases (ATP → ADP) * These residues are de-phosphorylated by protein phosphatases (H2O → Pi, hydrolyzes off phosphate)

Answer 69

* Glycogen phosphorylase * Breaks off one monomer of glucose (from glycogen) to glucose-1-phosphate * Very tightly regulated by allosteric inhibition and phosphorylation

Answer 70

* Inactive dimer, AMP is allosteric activator, ATP and G6P are allosteric inhibitors * Phosphorylase kinase activates, phosphoprotein phosphatase deactivates

Answer 71

1. Adrenaline binds to receptors on the cell surface - GPCRs 2. GPCRs produce CAMP 3. CAMP activates protein kinase A (PKA) 4. PKA phosphorylates phosphorylase kinase (activating it) 5. Phosphorylase kinase phosphorylates glycogen phosphorylase (activating it) 6. Glycogen is broken down 7. Glucose and ATP concentrations rise, inhibiting glycogen phosphorylase

Answer 72

1. Identify protein target 2. Establish activity assay - fluorescent/UV-vis readout 3. High throughput inhibitor screen 4. Identify lead compound 5. Optimize compound 6. Arrive at a potent, selective inhibitor 7. Animal studies (pharmacokinetics) 8. Human studies

Answer 73

* 96-well plate with different compounds * Want an enzyme inhibitor that decreases the amount of product formed

Answer 74

* Structure base drug design - get crystal structure of protein bound to the compound, what groups can you add to the compound to make it bind better to its target

Answer 75

* Structure base drug design - get crystal structure of protein bound to the compound, what groups can you add to the compound to make it bind better to its target

Answer 76

* potent - how well does it bind to its target * selective - does it bind to other cellular targets

Answer 77

* stability in vivo * oral bioavailability * metabolism * excreted by kidneys

Answer 78

* Phase I: safety (20-100) healthy individuals * Phase II: efficacy (100-500) diseased vs placebo * Phase III: long term safety/efficacy in larger population * FDA approval: phase IV - still collecting data

Answer 79

* cytochrome p450s - humans have 50-60 * they metabolize drugs * they are mono-oxygenases - have heme center * cytochrome p450s hydroxylate your drug * drug-drug interactions - one drug takes away a p450 that binds to another, making the level of the second higher * Pfizer Mpro inhibitor - combination therapy * add HIV drug to prevent drug metabolism

Answer 80

* Aka Michaelis-Menten kinetics * concentration of substrate is much greater than concentration of enzyme, concentration of enzyme substrate complex stays constant with time * therefore, rate of the reaction is proportional to the concentration of [ES]

Answer 81

* Michaelis-Menten Equation describes the relationship between rate and [S]

Answer 82

* V_max= k₂[E_T] (when enzyme is saturated w substrate) * K_m is the concentration of substrate at which V = (V_max/2)

Answer 83

* Michaelis Constant * Represents the affinity of an enzyme for a substrate * Lower K_m = higher rate at lower [S] → more efficient catalysis * K_m is unique for each enzyme substrate pair

Answer 84

* (K_cat) / (K_m) = catalytic efficiency → allows you to compare two different enzymes * the most efficient enzymes have a catalytic efficiency = 10⁸- 10⁹M^-1s^-1 * this is known as the diffusion controlled limit * Eg. acetylcholinesterase, and catalase (breaks down hydrogen peroxide)