Week 4

Question 1

What are the three types of reversible inhibition?

Answer 1

Competitive, uncompetitive, noncompetitive

Question 2

What is competitive inhibition?

Answer 2

Inhibitor competes with the substrate for the active site

Question 3

What is uncompetitive inhibition?

Answer 3

Inhibitor binds to the enzyme-substrate, preventing product

Question 4

What is noncompetitive inhibition?

Answer 4

The inhibitor binds to the allosteric site, changing the active site so the substrate isn’t able to bind

Question 5

What does it mean for inhibition to be reversible?

Answer 5

The inhibitor binds then releases

Question 6

What happens to the Km and Vmax during competitive inhibition?

Answer 6

Km increases, Vmax doesn’t change

Question 7

What happens to the Km and Vmax during uncompetitive inhibition?

Answer 7

Km lowers, Vmax lowers

Question 8

What happens to the Km and Vmax during noncompetitive inhibition?

Answer 8

Km doesn’t change, Vmax lowers

Question 9

What is the effect of increased Km on binding?

Answer 9

Binding isn’t as good

Question 10

In what situation is product formed during uncompetitive inhibition?

Answer 10

When the enzyme and substrate are bound, before the inhibitor binds

Question 11

During non-competitive inhibition, what happens to the Km when the inhibitor molecule binds to the enzyme? When the substrate binds after?

Answer 11

Km increases, substrate binding decreases. Km decreases, substrate binding enhances

Question 12

What happens when Vmax increases?

Answer 12

Rate of product formation increases

Question 13

What does the lineweaker-burk plot look like for competitive inhibition?

Answer 13

Question 14

What does the lineweaker-burk plot look like for uncompetitive inhibition?

Answer 14

Question 15

What does the lineweaker-burk plot look like for noncompetitive inhibition?

Answer 15

Question 16

In competitive inhibition, why doesn’t the Vmax change?

Answer 16

It can be overcome by a sufficiently high concentration of substrate

Question 17

Identify competitive, noncompetitive, and uncompetitive inhibition from the Michaelis-Menten plot

Answer 17

Question 18

What are the key features of irreversible inhibitors?

Answer 18

• Bind very tightly to active site
• No product forms, shuts the enzyme down
• Elucidates the mechanisms of enzyme action by covalently bonding to the enzyme
• Group-specific reagents react with R groups of specific amino acids

Question 19

What are the 4 catalytic strategies used by enzymes?

Answer 19

1. Covalent catalysis - Active site contains nucleophile that covalently binds
2. Acid-base - Molecule other than H2O donates or accepts a proton
3. Metal Ion - Funciton in several ways, serves as an electrophilic catalyst
4. Approximation and orientation - Brings two substrates together in an orientation that facilitates catalysis

Question 20

Enzyme activity can be modulated by?

Answer 20

Temperature, pH, and inhibitory molecules

Question 21

Explain the chymotrpsin mechanism

Answer 21

1. Lower barrier H-bond stabilizes His so that it can deprotonate Ser.
2. Once H+ is removed, Ser can act as a stronger nucleophile to attack electrophilic amide C.
3. Tetrahedral oxyanion is stabilized by H-bonding interactions with the oxyanion hole
4. Collapse of the tetrahedral intermediate and H+ transfer from His leads to the cleavage of the C-N bond. The N-terminal peptide is bound through acyl linkage to Ser
5. A water molecule binds to the active site and attacks the acyl ester carbonyl
6. The resulting 2nd oxyanion intermediate tetrahedral is stabilized via enthalpic interations with the oxyanion hole
7. The N-terminal fragment is released and returns to its initial state

Question 22

What is the purpose of the oxyanion hole?

Answer 22

Stabilizes the negative charge of oxygen atom by making H-bond w/ amine side chaines of ser & gly

Question 23

What are the key features of myoglobin?

Answer 23

• 8 subunits
• Globular tertiary structure
• Hydrophobic part of heme buried in the center of the protein
• Hydrophilic part of heme exposed to the surface

Question 24

What are the key features of the catalytic triad?

Answer 24

• Asp 102 stabilizes His
• His 57 pulls H from Ser
• Ser 195 acts as a nucleophile

Question 25

What is Heme?

Answer 25

Prosthetic group which is a precursor to hemoglobin, which is necessary to bind oxygen in the bloodstream

Question 26

Identify the key components of the heme

Answer 26

Question 27

In a heme, how is the Fe held?

Answer 27

6 coordinate covalent bonds from the four nitrogens in the tetrapyrrole group

Question 28

What are the key features of the 4th, 5th, and 6th coordination sites of Fe of a heme?

Answer 28

- 4th is occupied by nitrogens in the tetrapyrrole ring - 5th is nitrogen from proximal histidine - 6th is occupied by O2

Question 29

Identify the distal, O2, and proximal section of the histidine

Answer 29

Question 30

What is the key feature of the Nitrogen of the distal histidine?

Answer 30

It's too far away to participate in coordinate covalent bonding

Question 31

What is the function of the hydrogen attached to the N of the distal histidine?

Answer 31

Stabilizes O2 when it's present and bound to Fe²⁺

Question 32

When O2 isn't present, what does the distal histidine do?

Answer 32

Protects Fe²⁺ from oxidation

Question 33

What is oxidation?

Answer 33

Loss of an electron

Question 34

What happens when Fe²⁺oxidizes in the distal histidine?

Answer 34

Protein denatures, cannot reversibly bind O2

Question 35

What is the dissociation constant?

Answer 35

K_d, quantitative measure of the affinity of a ligand for its protein

Question 36

What is the chemical reaction between myoglobin and oxygen?

Answer 36

Mb + O₂ -\> MbO₂

Question 37

What is the formula for the dissociation constant for myoglobin?

Answer 37

K_d = [Mb][O₂]/[MbO2]

Question 38

What is fractional saturation?

Answer 38

Y, proportion of myoglobin molecules that have bound to O₂

Question 39

What is the equation of fractional saturation for myoglobin?

Answer 39

Question 40

What is K_d when Y=1/2?

Answer 40

K_d = O₂

Question 41

What happens to the ligand when K_d decreases? Why?

Answer 41

Greater affinity for its protein. As K_m decreases, the greater the affinity of substrate for enzyme

Question 42

When comparing hemoglobin to myoglobin, which has a higher affinity for oxygen?

Answer 42

Myoglobin

Question 43

What is a key feature of hemoglobin?

Answer 43

It's allosteric, has multiple subunits (quaternary structure)

Question 44

What is the equation of fractional saturation (Y) for hemoglobin?

Answer 44

Question 45

What is the relationship between the hill coefficient and cooperativity?

Answer 45

Bigger Hill coefficient, more cooperativity

Question 46

What does cooperativity mean in regards to hemoglobin?

Answer 46

If one heme group in hemoglobin has a bound O₂, it converts hemoglobin to a high-O₂ affinity state, increasing the ability of the other heme groups to bind O₂

Question 47

What is a key feature of hemoglobins AA sequences?

Answer 47

They're similar but not identical

Question 48

Hemoglobin has how many subunits, alpha, and beta chains?

Answer 48

- 4 subunits (tetramer) - 2 alpha chains, 2 beta chains

Question 49

What are the differences between myoglobin and hemoglobin?

Answer 49

* 18% similar in primary structure * Very similar in secondary and tertiary structure with 8 alpha helices * Hemoglobin can undergo quaternary structure * Common evolutionary origin

Question 50

What's the difference in structure of hemoglobin going from deoxyhemoglobin to oxyhemoglobin?

Answer 50

Iron gets smaller because it now shares electron density with oxygen and it's pulled further up into the plane of ring making the binding energy more favorable

Question 51

What happens during the formation of oxyhemoglobin in addition to Fe pulling up into the plane?

Answer 51

O₂ pulls up the proximal histidine which occupies the 5th coordination site causing a conformational change in the quaternary structure

Question 52

What's another way to describe the transformaiton of deoxyhemoglobin to oxyhemoglobin?

Answer 52

T-state to R-state

Question 53

Explain the respiratory system in regards to deoxyhemoglobin and oxyhemoglobin

Answer 53

1. O₂ comes in through the lungs into the arteries 2. Hemoglobin transports O₂ to the tissues where it's used for aerobic metabolism in the mitochondria 3. Dissolved O₂ diffuses freely or is bound to myoglobin, aiding in the transport of O₂ to the mitochondria 4. CO₂ is carried back to the lungs by hemoglobin or in plasma and is then released

Question 54

How does the binding of 2,3-BPG affect the T state?

Answer 54

Stabilizes it, lowers binding affinity for O₂ allowing hemoglobin to offload O₂ into cells

Question 55

What are the key features of 2,3 biphosphoglycerate?

Answer 55

* Small * Highly charged * Size of hole in deoxyhemoglobin is the perfect size for it and is lined with positively charged residues

Question 56

What is the Bohr effect?

Answer 56

Stimulation of oxygen release by carbon dioxide and H⁺

Question 57

What are the key features of the Bohr effect?

Answer 57

* CO₂ easily moves across membranes (small, nonpolar) * CO₂ becomes an acid in H₂O which then undergoes acid-base equilibrium to produce H⁺

Question 58

What is the relationship between pH, H⁺, and O₂

Answer 58

-, +, more O₂ will be released

Question 59

Lowered pH allows the formation of what in regards to hemoglobin?

Answer 59

Formation of ionic interactions stabilizes the T-state, enhancing O₂ release

Question 60

What is the relationship between CO₂ and hemoglobin?

Answer 60

CO₂ reacts with terminal amino groups to form negatively charged carbanate groups which form salt bridges, stabilizing the T-state

Question 61

What are CO₂ and H⁺ to O₂?

Answer 61

Heterotropic regulators, binding by Hb

Question 62

Describe the relationship of pCO₂, H⁺, pO_2, and O₂ in both tissues and lung

Answer 62

In tissues: +,+,-, oxygen is released In lungs: -,-,+, oxygen is acquired

Question 63

In what form is CO₂ transported from tissue to lungs?

Answer 63

Bicarbonate

Question 64

How does CO₂ get transformed into bicarbonate?

Answer 64

Carbonic anhydrase

Question 65

What is the function of allosteric effectors?

Answer 65

Bind to their target proteins and promote a conformational change that changes the functional properties of the protein

Question 66

What is the diffference between homotropic allosteric and heterotropic allosteric effectors?

Answer 66

Homotropic ae's bind at the active site whereas heterotropic ae's bind at other sites on the protein

Question 67

What are the positive and negative effectors for hemoglobin?

Answer 67

* O₂ is a positive homotropic effector, increasing the binding affinity of O₂ to hemoglobin * H⁺, CO₂, 2-3 BPG are negative heterotropic effectors, decreasing binding affinity

Question 68

What is HbA, HbS, and HbF?

Answer 68

HbA is normal adult Hb HbS - sickle cell HbF - fetal Hb

Question 69

What causes sickle cell?

Answer 69

Point mutation, AA, on beta chain

Question 70

Where does HbF lie on the fractional saturation vs. pO₂ graph? What does that mean?

Answer 70

In between myoglobin and hemoglobin. HbF has a higher affinity for O₂ than Hb, but lower than myoglobin

Question 71

What occurs during carbon monoxide poisioning?

Answer 71

CO is a competitive inhibitor, leading to increased O₂ affinity on the other subunits, so Hb will not release enough O₂