M114 Final

Explain phototransduction cascade

1. Photon hits rhodopsin and excites retinal

2. 11-cis retinal isomerizes to all-trans-retinal

3. Rhodopsin now bleached → all-trans-retinal and opsin separate

4. Enzyme burns ATP to convert all-trans-retinal back to 11-cis-retinal

5. Opsin and 11-cis-retinal reassociate

How/where does retinal attach to rhodopsin

retinal is covalently attached to Lys 216 on opsin through Schiff base

How is absorptive wavelength of a specific molecule defined

difference in energy between pi and pi* energy states

Blue shift vs. red shift

blue shift → short wavelength, high energy, occurs in molecules with less conjugation

red shift → long wavelength, low energy, occurs in molecules with more conjugation

Effect of more conjugation on absorptivity and reasoning

more conjugation leads to red shift (absorption at lower energy) because energy difference between pi and pi* decreases → less energy needed to excite photon

What is the extinction coefficient

epsilon: how efficient a substance absorbs light at a specific wavelength

How do we see 3 colors with only one retinal

we have 3 opsins that bind retinal in slightly different conformations, affecting the conjugation of retinal, shifting its absorption wavelength

Types of cones

red (L, long wavelength)

green (M, medium wavelength)

blue (S, short wavelength)

How can bees see UV light

bees have retinal that uses a deprotonated Schiff base, which allows them to absorb shorter wavelengths (higher energy) than humans

Relationship between transmittance and absorbance

A = -log(T)

T = I/I0

Beer’s Law

A = Ecl

Dynamic range of UV/Vis spectrophotometer and why

0.05-2

low absorbance means insignificant difference in comparison of I and I0

high absorbance means a very small amount of light is being transmitted to measure

How does conjugation affect extinction coefficient

more conjugation → higher extinction coefficient, ex. AMP & GMP have higher extinction coefficient @ 260 nm because of 2 rings

What groups are and are not used to quantify protein amount and why

amide not used because their absorbance @ 180-230 nm depends on secondary structure (don’t know amounts)

aromatic side chains used because they absorb strongly @ 280 nm (less overlap with other signals)

Why does DNA contamination affect protein absorbance readings but protein contamination not affect DNA absorbance readings

DNA absorbs strongly @ 260-280 nm → small amounts of DNA affect protein absorbance readings @ 280 nm

Protein absorbs weakly @ 260 nm → small amounts won’t affect strong DNA signal @ 260 nm

What is optical density

measure of light scattering at some wavelength to monitor cell growth

What is Stokes shift

difference between wavelength of absorbance and fluorescence

Fluorescence anisotropy measures

the motion of a fluorophore

How does FRET work

donor fluorophore emits light at a wavelength that can excite acceptor fluorophore

If donor and acceptor within ~10 nm, energy transfer occurs and acceptor emits light (FRET signal)

What does circular dichroism measure and how

measures secondary structure by detecting difference in absorption of circularly polarized light by amide bonds in chiral protein

Alpha helix CD characteristics

pi → pi* is positive

n → pi* is negative (double trough)

pi → pi* (+) is double the intensity of n → pi* (-)

Beta sheet CD characteristics

pi → pi* is positive

n → pi* is negative

pi → pi* (+) is similar intensity of n → pi* (-)

Random coil CD characteristics

pi → pi* is negative

n → pi* is positive

Criteria for metabolic pathway

individual reactions must be specific and pathway in total must be thermodynamically favorable

Purpose of adenine in ATP

acts as a “handle” that enzymes can hold on to to hydrolyze ATP

Why is ATP hydrolysis energetically favorable

1. Relieves the electrostatic repulsion between the 3 phosphate groups → decreases net negative charge

2. Pi is more stabilized by resonance than ATP

3. Increase in entropy

4. ADP and Pi are better solvated by water

Why is PEP a high energy molecule

1. High charge density

2. Hydrolysis products are stable → pyruvate and Pi (pyruvate can tautomerize into its keto form from its enol form)

Why is phosphocreatine useful

it stores high energy phosphate groups and can quickly donate its phosphate to ADP to regenerate ATP

How do cells make phosphocreatine if it’s a high energy molecule

1. Cells compartmentalize the phosphorylation of creatine in the IMS, where [ATP] and [creatine] are high

2. Cells remove product (phosphocreatine) as it’s formed to drive the reaction forward

Steps of phosphocreatine formation/use

1. ATP transported into IMS from matrix, creatine transported into IMS from cytosol

2. Creatine reacts with ATP via mitochondrial creatine kinase to make phosphocreatine

3. Product removed into cytosol

4. Phosphocreatine can now react with ADP via cytoplasmic creatine kinase to regenerate ATP

Isozyme of creatinine kinase in blood can indicate

heart attack

High levels of creatinine in blood can indicate

liver failure

1st order integrated rate law axes and slope

time vs. ln[A]

slope is -k

2nd order integrated rate law axes and slope

time vs. 1/[A]

slope is +k

What is half-life

time required for a reactant’s concentration to be ½ its initial value

How to find half-life

t1/2 = 0.693/k

What is isosbestic point

specific wavelength at which the absorbance of a mixture stays the same because reactant and product have the same molar absorptivity at that exact wavelength

What if no isosbestic point is seen

reaction is not 1st order–there is a third species involved

What is transition state theory

says that the rate of a reaction depends on how difficult it is for molecules to reach TS

Why does the rate constant change when a catalyst is added

because k depends on Ea (Arrhenius equation), and Ea decreases when a catalyst is added

What would happen if catalysts stabilized substrate rather than TS

the ES complex would be lower in energy than free S, making Ea even higher → ultimately slowing the reaction down

What is the 1.4 rule

every 10 fold change in rate (x10) corresponds to a 1.36 kcal/mol change in TS energy at room temp

Besides 1.4 rule, how can I find the change in rate

∆k = e^(∆∆Ea*/RT)

Why are saturating substrate conditions used

at high [S], almost all enzyme is in ES form → then k1 and k-1 are negligible and k2 becomes rate limiting

What is the steady state assumption

change in [ES] over time = 0, k2 ~ kcat

At what conditions does Vmax occur

at high [S] when enzyme is saturated and entirely in ES form

Km is…

the [S] where the velocity of the reaction is 1/2 Vmax and reflects apparent affinity of enzyme for its substrate

Lineweaver-Burk intercepts and slope

x-int is -1/Km

y-int is 1/Vmax

slope is Km/Vmax

What is kcat

turnover number of an enzyme: max number of substrate molecules enzyme can convert into product per unit time when fully saturated

What does large kcat mean

What does small kcat mean

large kcat → enzyme is efficient at converting substrate to product

small kcat → enzyme is not efficient

What does large Km mean

What does small Km mean

large Km → high [S] to reach 1/2Vmax, low affinity for enzyme

small Km → low [S] to reach 1/2Vmax, high affinity for enzyme

Competitive inhibition def, Vmax, kcat, Km

I competes with S for active site

no change in Vmax because high [S] can outcompete I

no change in kcat because I doesn’t affect ES → P + E when S DOES bind (turnover)

increase in Km because higher [S] will be needed to reach 1/2Vmax

Uncompetitive inhibition def, Vmax, kcat, Km

I binds only to ES complex, not free E

decrease in Vmax because some ES is inactivated by I → less ES to form product

decrease in kcat because some of ES is inactive and cannot turnover S to P

decrease in Km because ESI formation pulls E + S → ES reaction to the right, making the E appear to have higher affinity for S

Noncompetitive inhibition def, Vmax, kcat, Km

binds to free E or ES complex at allosteric site

decrease in Vmax because I binding inactivates enzyme → decreases number of active enzymes

decrease in kcat because remaining active E turns over S more slowly

no change in Km because I is not competing with S for active site

What is KD

dissociation constant of ES complex

ES → E + S

KD = [E][S]/[ES]

What is EC50

concentration of a drug needed to produce 50% of its maximal effect

What is HbA1C and how is it formed

HbA1c is glycated hemoglobin

formed when open-chain glucose (aldehyde) reacts with hemoglobin in RBCs to form a Schiff base

What can affect HbA1C formation

1. Transport of glucose into RBC by insulin-dependent transporter

2. RBC generation

3. Glycolysis

4. RBC elimination

kgly =…

kg x Vmax/(kc + Km)

Oxidoreductase function

transfer electrons/H atoms

Transferase function

group transfer reactions

Hydrolase function

hydrolysis reactions

Lyase function

cleavage of C-C, C-O, C-N, or other bonds by elimination

Isomerase function

transfer of groups within molecules to yield isomeric forms

Ligase function

formation of C-C, C-S, C-O, and C-N bonds by condensation reactions

Amino acids that can be used as general acids or bases (8)

Asp, Arg, Lys, Cys, His, Ser, Tyr, Glu

His 12 and His 119 in general acid/base catalysis

His 12 acts as a proton acceptor → below pH 5.5, His 12 gets protonated and can’t act as a proton acceptor anymore

His 119 acts as a proton donor —> above pH 6.5, His 119 gets deprotonated and can’t act as a proton donor

How do enzymes stabilize a carbanion

1. Forming enolate

2. Forming enolate/enol stabilized by H-bonds

3. Converting carbonyls into Schiff bases

4. Using metal ions to neutralize and stabilize charges

Metalloenzymes vs. metal-activated enzymes

metalloenzymes tightly bind metals (Fe2+, Fe3+, Cu2+, Zn2+, Mn2+, Co3+)

metal-activated enzymes loosely bind metals (Na+, K+, Mg2+, Ca2+)

What is charge shielding

metals help neutralize charges

How does water ionization work

1. Imidazole coordinates Zn2+

2. Zn2+ coordinates water

3. H-bonding network forms

4. His is activated through a transfer of Hs through water network

How is PEP formed if it’s a high energy molecule

a thioester intermediate is used → relatively unstable (high energy) intermediate that is used to get to a high energy product

What is kB

Boltzmann constant: used to calculate how much thermal energy is available at a certain temp → (kB)(T)

How does kinetic isotope effect (KIE) work

tells us which atoms are involved in RDS

an atom is replaced with a heavier isotope–if the rate decreases, that bond is involved in RDS because heavier isotopes vibrate more slowly and make the bond harder to break

Difference between primary and secondary KIE

primary KIE occurs when isotopic bond is broken in RDS → largest change in rate

secondary KIE occurs when isotopic bond is near reactive center, only changes hybridization or geometry → smaller change in rate

What is the catalytic triad

Asp-His-Ser in serine proteases that acts as a proton donor system to deprotonate Ser and create a good nucleophile to attack the carbonyl on the substrate

Serine protease mechanism

1. Substrate binds in specificity pocket → positions substrate’s scissile bond near the catalytic triad

2. Catalytic triad relay system: Asp stabilizes His, His deprotonates Ser, O- on Ser attacks substrate carbonyl

3. Backbone H-bonding from NHs in oxyanion hole stabilize negative charge on oxygen in tetrahedral intermediate

How does DIPF work

DIPF covalently and irreversibly inhibits serine proteases

1. Asp stabilizes His

2. His deprotonates Ser-OH

3. Ser-O- attacks electrophilic phosphorus of DIPF

4. Pentacoordinate intermediate

5. F leaves as LG

6. Phospho-serine bond is extremely stable, cannot be hydrolyzed → enzyme is permanently inactive

How does sarin gas work

sarin gas enters bloodstream and acts as a suicide inhibitor, covalently and irreversibly binds to acetylcholineesterase, forming a stable phosphorylated enzyme that cannot be hydrolyzed

Acetylcholine can no longer bind to acetylcholineesterase to be broken down, leading to muscle spasms

rhodopsin holding retinal

retinal (all-trans)

extinction coefficients of Trp, Tyr, Phe

Trp: 5250 M-1cm-1

Tyr: 1200 M-1cm-1

Phe: 190 M-1cm-1

adenine

guanine

cytosine

thymine

how does denaturation affect DNA’s UV absorbance

UV absorbance increases when denatured because electronic interactions between neighboring bases are disturbed

how does protein unfolding affect emission

fluorescence intensity decreases when exposed to solvent when unfolded

black: mostly alpha

red: mostly beta

green: mixture of alpha and beta

blue: random coil

hydrolysis of PEP and keto-enol tautomerization of pyruvate

competitive inhibition

competitive inhibition

uncompetitive inhibition

uncompetitive inhibition

noncompetitive inhibition

noncompetitive inhibition

hemoglobin and open-chain glucose

HbA1C

oxidoreductase

transferase