Ch.4 Protein Structure and Function III

Question 1

what is the first step in any enzymatic reaction

what are the 2 important sites

Answer 1

binding of substrate to ligand (models include hand in glove and induced fit)

• ligand binding site and the catalytic site are important but they may be the same site
• active site is the true catalytic site and is important

Question 2

where is the active site typically in an enzyme and why

Answer 2

-usually in a crevice or cavity to keep away from water molecules that can form H bonds and compete for ligand binding

Question 3

protein folding allows for what 2 things

Answer 3

• formation of active site

- clustering of aa side chains/ R groups for necessary non covalent interactions

Question 4

Define a catalyst and give the 3 types

Answer 4

increase the rate of reaction by making it more energetically favorable

1) brings together substrate and ligand in close proximity
2) enzyme strains the substrate to force it to transition state
3) binding of substrate to enzyme rearranges electrons in the substrate to create a partial neg and partial positive charge that favors a reaction

Question 5

describe allosterism (3)

Answer 5

• ligands bind to regulatory site of the enzyme and influence enzyme activity
• usually conformational change
• alters the active site

Question 6

describe positive vs negative allosterism

Answer 6

in positive:
regulatory ligand binds to the allosteric site to induce binding in a positive way/triggers “on”

in negative:
regulatory ligand binds to all. site to stop the binding to the substrate/ triggers “off” conformational change inhibits binding of substrate and enzyme

Question 7

describe the CTP model of negative feedback and T vs R state

Answer 7

• when lots of CTP is in the cell, it binds to the regulatory subunits of the enzyme
• binding forces enzyme into T state (tense) which is where the active site is blocked

-when less CTP is in the cell, it doesn’t bind to the regulatory site therefore the active site/ substrate binding sites are open and in a ‘relaxed’ state

Question 8

differentiate hemoglobin and myoglobin

where does oxygen bind?

Answer 8

myoglobin:

• 1 heme group
• higher affinity for oxygen than Hg
• carries oxygen in the muscle
• R groups can’t form quaternary so monomer and tertiary structure!!!!

hemoglobin:
-4 heme groups
-carries oxygen in the blood
-allosterically regulated 
-4 subunits: 2 alpha and 2 beta 
transports co2 and hydrogen 
*R groups on surface and can form quaternary 

*oxygen binds to heme= non polypeptide/prosthetic group

Question 9

where specifically is the heme group located?

how is it that the iron in the Hg doesn’t get oxidized? what is it called when it is oxidized?

Answer 9

• iron is bound to a histidine group so that the other electrons can be given to oxygen.
• if the electrons weren’t busy with histidine, then it would be oxidized and called methemoglobin
• heme group is located in a crevice within the globin molecule

Question 10

how would you plot myoglobin and hemoglobin to analyze?

what are the characteristic of the slope for both myo and hg?

what is p50?

Answer 10

plot the fraction of protein with bound O2 (fractional saturation) vs the concentration of O2 (partial pressure, p)

• myoglobin: hyperbolic line that reflects high affinity for 02. important to look at what myoglobin is doing in the TISSUES
• hemoglobin: sigmoidal curve (S) that reflects the average affinity of the 4 subunits for O2 binding
• p50 is the partial pressure of oxygen where 50% of the protein is saturated

Question 11

why is it important that hemoglobin has a sigmoidal curve?

Answer 11

at low concentrations of O2, binding is inhibited which is good because you want the O2 to stay in the tissues
and
you want the affinity to be highest in the lungs when you need O2 to bind to Hg

Question 12

describe characteristics of fetal hemoglobin

which way does the curve shift?

how come sometimes we don’t know baby has a blood disorder until 6mths of age?

Answer 12

fetal hg has higher affinity for oxygen (need to take it from mother) and has different Hg subunits

curve shifts to the left showing higher affinity for oxygen

-baby has gamma and beta subunits which as the beta develops the gamma diminishes so we have that period where we have both and we can’t tell

Question 13

hills coefficient proves what?

Answer 13

that the myglobin does not show allosterism/ positive cooperative binding like Hg does because it is a monomer.

n=1.0 for myoglobin and hg= 2.8
over 1 shows cooperative binding

Question 14

what are the 2 states for hemoglobin?
describe the shapes as well
when do we switch from one state to the other?

Answer 14

R: oxygen affinity is high. here we are binding :)
iron moves into the plane of the porphyrin ring, pulling histidine side chain in the same direction

*movement of histidine causes rearrangement of the helices and a conformational change that moves it away from its partner in the alpha-beta pair

T: oxygen affinity is low, we are not binding
The heme iron is slightly out of the plane

Switch from T to R when the first oxygen binds

Question 15

if there was no cooperatively binding, what would happen? note!

Answer 15

Hg would not be able to take up as much oxygen in the lungs
*note : there is little effect in the tissues
SO most of the Hg in the lungs is in the R state and in tissues its in the T state

Question 16

what happens to the channels in each state?

Answer 16

-in the T state, the channel is wider
in the R state, the channel is narrower

think of it as the channel wants to keep the hg in so it gets narrow when its bound

Question 17

how is oxygen binding regulated in Hg through DPG or 2,3 biphosphoglycerate? what is this an intermediate of ?

Answer 17

• intermediate of glycolysis (cell respiration)
• DPG cannot bind to Hg when it is oxygenated and goes to act as an intermediate in the cell respiration
• when hg is deoxygenated, DPG comes and binds bc it has nothing else to do ;) and ultimately its bc the channel is bigger

Question 18

what affect does DPG/BPG binding to DeoxygHb have on oxygen binding?

-what the difference btwn having and not having DPG?

Answer 18

=stabilizing affects that pushes it to T state and enhances oxygen release at the peripheral tissues (factor of 26 difference when you have BPG)

-stabilizes the T STATE

• so here we are going form R–T in the tissues
• DPG creates a competition for Oxygen binding, therefore it helps especially during exercise or high altitudes

Question 19

__ ph enhances the release of O2 from hg
increasing __ _also lowers oxygen affinity

what is the bohr effect?

Answer 19

low pH enhances the release of O2 from Hg

increasing CO2 lowers affinity for oxygen

Bohr:
Hb02 + H+ + C02—-> HbCo2H+ + O2
favors release of oxygen in metabolically active tissues
bc in metabolically active tissues you have low pH (lots of H) and high Co2

Question 20

binding of Co2 shifts the curve which way?

Answer 20

right

Question 21

what conformational changes occur during oxygen binding that are related to the Bohr effect?

Answer 21

Because histidine is so sensitive to protons, going from T to R state (deoxy to oxy) results in lower affinity for protons

• going from R to T increases affinity for protons so deoxyHb picks up protons as it goes through the tissues
• in T state his 146 is bonded to asp 94. the shift to the R state disrupts the H bond, allowing the proton on His to dissociate

Question 22

most CO2 is transported as___ and reacts ___ with ___

Answer 22

bicarbonate

CO2 + H20—> H2CO3—> HCO3 + H+

carbon dioxide reacts reversible with aa side chains of hb, helping to further stabilize the T state

Question 23

uptake of H by Hb has what 3 effects?

Answer 23

• stabilizes the T state
• assist in buffering tissues
• release of H in the lungs to get rid of acidity bc high concentration of oxygen promotes transition to the R state

Question 24

R state is stabilized by

Answer 24

oxygen, high pH , low Co2

Question 25

T state is stabilized by

Answer 25

high Co2, low pH, BPG

Question 26

affinity for protons and oxygen for T state vs R state?

Answer 26

T state has higher affinity for protons but lower affinity for oxygen

R state has low affinity for protons, but high affinity for oxygen

Question 27

A27-year-oldfirefighterisbroughttothe emergency room after being exposed to smoke during a training exercise. He looks ill and has labored breathing. He is clutching his head and exhibits an altered mental status. On examina- tion, you note that he appears red, and his pulse oximetry reads 100%. You suspect carbon monoxide toxicity. What is true of the oxygen saturation curve during carbon monoxide toxicity?

Answer 27

Oxygensaturationcurves relate the saturationofhemoglobinwithoxygen fora given partial pressure of oxygen. If carbon monoxide binds to one of the subunits of hemoglobin, the affinity of the other subunits for oxygen is increased (due to the cooperative nature of oxygen binding to hemoglobin). This shifts the oxygen binding curve to the left. Because the oxygen now has a higher affinity for hemoglobin, it is more difficult for hemoglobin to release oxygen to the
tissues, leading to hypoxia despite oxygen being bound to hemoglobin. Conditions that shift the curve to the right allow oxygen to be released more readily: low pH, increased PCO2, increased temperature, presence of 2,3-bisphosphoglycerate, and absence of carbon monoxide. In other words, hemoglobin will release oxygen in states that allow for normal binding of oxygen and increased oxygen demands by tissues.