Lecture 10

Question 1

What are some of the features of the serine protease catalytic mechanism that contributes to lowering of the activation nrg. Contributes to inc in rate of rxn?

Answer 1

Enzyme catalysis (Serine Protease Mechanism)

1. Binding site (binds substrate in specific way. Inc rate by increase local effective concentration). Maybe 2 substrates have to come together and keeping them there long enough for them to react. Having them in local effective conc can help guide fxn forward. Important for specificity. Enzymes don’t only inc catalytic rate but eliminate a lot of the side products u can get. Specificity for substrate. What kind of features of the substrate are recognized? Electrical stat/ shape (steric)/ stereochemistry/ polar
2. Orbital orientation (getting reactants to be in right juxtaposition). The 1st stage of the rxn in the orientation of the nucleophile the 2nd stage is orientation of second nucleophile (the water). (Ser/H2O). Certain angle, origination, and distance that is important for the nucleophilic attack. This is called the Burgi angle (angle of attack on carbonyl of nucleophile).
3. Shifting of pKa values. (e.g. weird enzyme not as histidine as general base
4. Electrostatic stabilization (TS stabilization)
5. Covalent catalysis (not all enzymes have this but directly brings substrate to different pathway)

Question 2

What does the hill coefficient (n) describe?

Answer 2

Cooperativity; it describes the fraction of the macromolecule saturated by ligand as a fxn of ligand conc

Question 3

What does
n = 1
n > 1
n < 1

mean?

Answer 3

1. n=1 (no cooperativity) completely independent binding, regardless of how many additional ligands are already bound
2. n>1 (+ cooperativity) Once one LIGAND molecule is BOUND to the PROTEIN, its AFFINITY for other ligand molecules INCREASES.
3. n<1 ( - cooperativity) Once one ligand molecule is bound to the LIGAND, its affinity for other ligand molecules DECREASES.

Question 4

How is myoglobin structurally related and functionally related? How do they differ in terms of cooperativity

Answer 4

Myoglobin - monomer, no cooperativity
Hemoglobin - oligomer, cooperativity

Functionally related cause bind oxygen

Question 5

What is the eqn for the reversible binding of O2 to myoglobin?

Answer 5

Mb(O2) —->

Question 6

What is Y for Mb?

Answer 6

Fractional saturation of Mb w/O2

Question 7

Eqn for Y for Mb?

Answer 7

Y = [MbO2] / [MbO2] + [Mb]

Question 8

Draw Y vs. pO2 plot and hill plot

Answer 8

Slide 4

Question 9

What is the eqn for the reversible binding of O2 to the 4 sites of Hb?

Answer 9

Hb(O2)4 —->

Question 10

What is Y?

Answer 10

Fractional saturation of Hb w/O2

Question 11

What is eqn for Y for Hb?

Answer 11

Y = [Hb(O2)4] / [Hb(O2)4 + [Hb]

Question 12

Draw Y plot for Hb and Hill plot

Answer 12

Slide 13

Question 13

Organic part of Heme?
____________

What type of iron?
____________

What type of cofactor?
_____________

Geometry of coordination?
\_\_\_\_\_\_\_\_\_\_
\_\_\_\_\_\_\_\_\_\_
\_\_\_\_\_\_\_\_\_\_
\_\_\_\_\_\_\_\_\_\_

Answer 13

Organic part of Heme?
1. Porphyrin ring

What type of iron?
Fe II (Fe2+) (Ferrous)

What type of cofactor?
Prosthetic group

Geometry of coordination?

1. Octahedral
2. Tetragonal bipyramidal
3. 5 N ligands
4. 1 O ligand

Question 14

Slide 7

Answer 14

N/A

Question 15

Compare Mb vs. Hb in terms of oxygen saturation

Answer 15

Slide 8

Question 16

Myoglobin

1. Fxn?
2. Found where?
3. How does O2 get in and out

Answer 16

1. Fxns to store O2
2. Found in muscle cells
   - Gives meat its red colour, when exposed to O2
   - deoxymyoglobin is PURPLE
3. O2 gets in and out due to breathing (dynamics) of the protein

Question 17

What is the most abundant protein in mammals?

Answer 17

Hb

Question 18

Where is Hb located?

Answer 18

Erythrocyte cytosol

Question 19

Describe the 3-D structure of Hb

Answer 19

1. 2 α/β protomers
2. A dimer of heterodimers
3. 4 heme, 4 O2 binding sites
4. Globin fold (globular) – all α protein fold

Question 20

Describe structure of deoxyHb

Answer 20

1. Water binds to Fe

2. Iron atom lays BELOW the PLANE of the HEME.

Question 21

How does the structure of Hb change when going from deoxyHb to oxyHb

Answer 21

In deoxyHb, water binds to Fe. The Fe atom lays below the plane of the heme.
In oxyHb, the binding of O2 pulls Fe into plane of the heme. This pulls on the PROXIMAL His, which pulls out of helix. This motion is then propagated thru the protein, leading to cooperative O2 binding.

Question 22

The cooperativity is achieved through domain rotation ~ ____ degree in _____ rotation

Answer 22

The cooperativity is achieved through domain rotation ~ 15 degree in protamer rotation

Question 23

What are the 4 other molecules that can bind to Hb besides O2?

Answer 23

1. nitric oxide (NO): cardiovascular signalling molecule.
2. carbon monoxide (CO): poison. Forms a stable complex hard to remove
3. carbon dioxide (CO2): product of respiration
4. 2,3-bisphosphoglycerate (2,3-BPG): binds to deoxygenated Hb promotes release of O2, tense state

Question 24

Slide 19

Answer 24

N/A

Question 25

What is the molecule called when CO2 binds to Hb

Answer 25

Carbaminohemoglobin

Question 26

How many molecules can Hb bind to of Co2?

Answer 26

1. Hemoglobin can bind to 4 molecules of CO2
2. The CO2 molecules form a carbamate with the 4 terminal-amine groups of the 4 protein chains in the deoxy form of the molecule.

Question 27

Draw eqn for carbamate molecule

Answer 27

Protein-NH2 + CO2

—->

Question 28

1. Where does 2,3-BPG bind to Hb?
2. How does this effect the affinity for O2?
3. How many molecule(s) of 2,3-BPG bind per Hb?

Answer 28

1. Binds to beta-subunits
   Near center
2. Stabilizes T state
   reduces oxygen affinity
3. 1 molecule binds per Hb

Question 29

2,3-BPG causes a _____ shift in the Hg-oxygen saturation curve

Answer 29

RIGHT