Lesson 7: Hemoglobin - Allosteric Modifiers Introduction to Enzymes

Question 1

positive allosteric effector: O2

Answer 1

positive because binding O2 to one site increases affinity at another site

Question 2

negative allosteric effectors: H+, BPG, and CO2

Answer 2

heterotropic negative allosteric modifiers — these all decrease a subunit’s affinity for O2

Question 3

what happens if we mutate 2 His residues in central cavity to Ala
(Hb)

Answer 3

if ala binds, means that 2,3 - BPG binds less (needs a basic, positive environment) … if you bind less of it it’ll shift to R state (graph pushed left)
- still cooperativity from the other oxygen binding
- between stripped Hb and Blood Hb

Question 4

what happens if we mutate 4 His residues in central canal to Ala?

Answer 4

same as 2 mutations, but even more so to the left
- N+ terminals will bind some BPG still, but the amount overall is reduced, more R dominant reaciton
- between stripped Hb and Blood Hb

Question 5

what if we kept 4 His residues and added 2 more Lys to the central cavity

Answer 5

Lys is basic –> adding more positive charge denity, more 2,3-BPG stabilization for T-state, in front of the Blood Hb

Question 6

where can the homodimer in the lecture show cooperative oxygen binding

Answer 6

salt bridges between N and C termini, multiple subunits

Question 7

will the homodimer exhibit a Bohr effect

Answer 7

yes - HIS 13 will function like HIS 146 in Hb
- deprotonate which increases oxygen bonding

Question 8

what is a bohr effect

Answer 8

changing O2 binding affinity by changing pH

Question 9

would the addition of BPG have an effect on O2 bindingt (to the central cavity that does not have a lot of positive charge density)

Answer 9

not likely – requires a LARGE amount of positive charge density in the central cavity

Question 10

what would be the effect of a mutation which replaced aspartic acid 85 with a lysine

Answer 10

most likely increase O2 binding
——- charge charge respulsion, cause the helix to spread apart and O2 can get into the canal

Question 11

how does CO2 modulate Hb binding affinity

Answer 11

through the HCO3- buffering and carbonic anhydrase

Question 12

CO2 buffering

Answer 12

• CO2 diffuses from the tissues to the rbc
• carbonic anhydrase causes rxn to quickly yield HCO3- and H+ (a majority of CO2 is carried through the vascular system in the form of HCO3-)
• since H+ is being released, it stimulates O2 to be released into the tissues

Question 13

CO2 alone helps shift () and () O2 transfer

Answer 13

R –> T and increase

Question 14

BPG alone helps shift () and () O2 transfer

Answer 14

R –> T and increase

Question 15

combined, BPG and CO2+….

Answer 15

most efficiently shift R–> T and increase O2 transfer

Question 16

Carbonic Anhydrase

Answer 16

CO2 + H2 <-> H2CO3 <-> H+ and HCO-
- increase CO2 means increase H+
- increase H+ shifts
R –> O2 dissociates

Question 17

CO2 decreases what

Answer 17

Hb affinity for O2 because of carbonic acid buffering sytem

Question 18

where are a lot of the pathological substitution mutations structured near

Answer 18

oxygen binding sites

Question 19

Hiroshima

Answer 19

• B146 (HC3)
• His –> Asp
• disrupts salt bridge in deoxy state

Question 20

Suresnes

Answer 20

• A141 (HC3)
• Arg –> His
• eliinates bond between Arg 141 and Asn 126 in deoxy state

Question 21

Hiroshima and suresnes both favor

Answer 21

both favor R state because they destabilize the T-state (“deoxy”)

Question 22

would ppl with Hb suresnes and Hb hiroshima be sigmoidal

Answer 22

yes – we know because they are still alive meaning that they must have some level of cooperactivity

Question 23

enzymes allow for

Answer 23

• increased reaction rates (10^3 - 10^19 times)
• “mild” reaction conditions (i.e. physiological, temp, pH)
• great specificity: both substrates consumed and products produced
• coordinate control: reactions can be turned on and off by modulating activity of enzyme

Question 24

1

Answer 24

oxidoreductase
- oxidation/reduction reactions
ex: oxidases/dehydrogenase

Question 25

2

Answer 25

transferace - transfer of functional groups ex: kinasdes, transaminases

Question 26

3

Answer 26

hydrolase - formation of 2 products by hydrolyzing a substrate ex: peptides, lipases

Question 27

4

Answer 27

lyase - cleavage of C-C, C-O, C-N and other bonds by means other than hydrolysis or oxidation ex: decarboxylases, carboxylases

Question 28

5

Answer 28

isomerase - intramolecular rearangements, transfer of groups within molecules ex: mutases, isomerases

Question 29

6

Answer 29

ligase - formation of C-C, C-O, C-S, or C-N bonds using ATP leverage ex: synthetases

Question 30

do enzymes change Keq

Answer 30

no - they cannot change delta G

Question 31

do enzymes alter the k of the reaction

Answer 31

yes!

Question 32

relationship between Keq and delta G

Answer 32

as Keq increases, delta G gets more Negative (think: spontaneous)

Question 33

where does the rate increase come from

Answer 33

the lower activation energy barrier

Question 34

what do catalysis to

Answer 34

lower the amount of energy required to reach the transition state (activation energy)

Question 35

is there a change in delta G when a catalyst use

Answer 35

no -- there is no change betwen the delta G of the ground state reactants and the ground state products

Question 36

comparison of rate enhancement by heat vs decrease in activation energy

Answer 36

- at high T, a large number of molecules possess the threshold energy to get over the activation energy barrier - at low T, only a small number of molecules possess the threshold energy to get over the activation energy barrier

Question 37

comparison of rate enhancement by heat vs decrease in activation energy continued...

Answer 37

- lowering activation energy increases the number of molecules that possess the threshold energy to get over the activation energy barrier

Question 38

what does lowering the value of delta G ++ do

Answer 38

increase the number of molecules with sufficient energy to attain the transition state

Question 39

proximity and orientation

Answer 39

- enzymes bine substrates with geometric and electrostatic complementary - bring reacting functional groups in close proximity for chemistry to occur - enzyme maximizes weak interactions between the E and S (aka energy that is released in the form of heat = binding energy) This binding energy propels the enthalpy driven conformational changes in the enzyme

Question 40

why is the chemistry rate limiting

Answer 40

highest activation energy in catalyzed reaction ES --> EP

Question 41

preferential transition state binding

Answer 41

- enzymes preferentially stabilize substrates at or near the transition state

Question 42

what would happen to delta G if the enzyme stabilized the substrate

Answer 42

the mazimum stabilization of S decreases energy of ES complex below energy of P -- very stable structure --> hard to form products - catalyzed activation energy barrier would be incredibly large , endergonic rxn in this model the catalyzed activation energy of delta G ++ cat, is the sum of the uncatalyzed rxn, delta G ++ uncat + contrimuted delta G magnetic interactions

Question 43

enzyme stabilizing the reaction state

Answer 43

- increases concentration of molecules in transition state - limits ES -> E + S increases rate of ES-> P

Question 44

how do we know that enzymes preferentially stabilize transition states

Answer 44

Transition State Analogs - stable molecules that mimic the proposed transition state - these molecules bind tightly to the active site

Question 45

enzyme adenosine deaminase

Answer 45

- binds the transition state analog 6-hydroxy-1,6-dihydropurine 10 x than substrate or prouce

Question 46

general acid base chemistry

Answer 46

- certain amino acids of the ability to perform acid-base chemistry

Question 47

catalytic functional groups

Answer 47

His, Asp, Glu, Lys, Arg ^^^^^ normal charged groups Ser, Thr ^^^^^^ possess OH Tyr, Cys

Question 48

covalent catalysis

Answer 48

- amino side chains form a transient covalent bond with substrate - this describes the specific, transient, covalent bond between E and S --> there will be other bonds being made or broken

Question 49

electrostatic catalyis

Answer 49

not acid/base - enzyme side chains, components of the peptide bond, and N- and C- termini, can stabilize charged intermediates ex: - His 57 R-group stabilizes lone pair on amide N through electrostatic interactions - Amide portin of enzyme Ser 195 and Zgly 193 stabilize oxyanion through electrostatic interactions

Question 50

metal ion catalysis

Answer 50

metal ions can stabilize charged groups, carry electrons, or promote S binding

Question 51

catalytic triad of chymotrypsin: Ser 195 and Adp 102 and His 57

Answer 51

Ser 195 and Asp 102 and His 57 establish H-bond network that facilitates reactivity - when the molecule folds -- these 3 R groups are right next to eachother.... H-bonding!

Question 52

chymotropsin:

Answer 52

hydrolyzes peptide bonds C-terminal to aromatics --- large substrate binding pocket accommodates aromatic residues such as tyrosine

Question 53

chymotropsin: step 1

Answer 53

polypeptide substrate binds to enzyme active site

Question 54

chymotropsin: step 2

Answer 54

Acid/base catalysis and covalent catalysis -- His 57 removes a proton from ser 195, which allows a nucleohilic attack by the serine oxygen on the carbonyl carbon of the peptide

Question 55

chymotropsin: step 3

Answer 55

ser 195 side chain: covalent bond with substrate carbonyl electrostatic stabilization of oxyanion

Question 56

chymotropsin: step 4

Answer 56

acid/base catlysis: His 57 acts as base electrostatic catalysis: amide protons stabilize oxyanion

Question 57

chymotropsin: step 5

Answer 57

acid/base catalysis: His 57 acts as an acid oxyanion "breaks down"

Question 58

chymotropsin: step 6

Answer 58

oxyanion "breaks down" yields product 2 (amino-terminal fragment) and funcitonal catalytic triad)

Question 59

role of Mg 2+

Answer 59

stabilizing (-) charge on carboxylate -- electrostaic stabilization