10-13: ALLOSTERY

Question 1

allostery definition

Answer 1

binding of one ligand to enzyme/protein is affected by the binding of another (effector or modulator) at different site

Question 2

homotropic and heterotropic effect definitions

Answer 2

homotropic effect: regulation is by ligands (deal w/substrate)

heterotropic effect: regulators are different from ligands (dealing w/ A or I; not the S-binding sites)

Question 3

tense vs relaxed state

Answer 3

T:

• affinity for inhibitor is strong so it is well bound
• dissociation is low
• S and activator (A) may not bind well

in chemical equilibrium with:
R:
-change affinity of binding of A, I, S
-overall structural change
-S is in pocket
-I does not fit well; A fits v. well

Question 4

feedback inhibition example definition

Answer 4

-heterotropic allosteric inhibition where final product of pathway is chemically unrelated to substrate, binds to BS which is not S-BS and then deactivates the enzyme chemical conversion

Question 5

ATCase enzyme (Aspartate Transcarbomyolase)

Answer 5

• e.g. heterotropic allosteric inhibition regulation
• ATCase catalyses the comitted step in pyrimidine biosynthesis; carbamoyl phosphate + aspartate = CTP (cytidine triphosphate) in many steps
• CTP is necessary for RNA synthesis
• bind ATCase in regulatory BS that affects enzyme activity of ATCase; signals that there’s enough CTP so will deactivate ATCase in cell so can stimulate CTP for production for RNA synthesis/transcription or can stimulate the sue of aspartate towards amino acid synthesis

Question 6

regulating ATCase

Answer 6

• controlled by feedback inhibition
• rate of product formation/enzymatic conversion of ATCase dependent on adding CTP not on S
• CTP does not fit into active site yet more we add, lower the rate

Question 7

ATCase structure

Answer 7

-composed of 3 regulatory dimers (heterotropic control) and 2 catalytic trimers (homotropic regulation)

Question 8

Identification of PALA as a competitive inhibitor of ATCase

Answer 8

• mimics the intermediate (looks similar but chemically stable)
• active site of ATCase contains residues from more than one subunits; found in between catalytic trimer
• in presence of competitive inhibitor, could have 100% of ATCase in active conformation and fully occupy BS (not possible w/ S because will be converted)
• the direction of the individual subunits that forms a high affinity BS for S with contributions from amino acids in both subunits

Question 9

structural effect upon binding of PALA

Answer 9

• T to R state
• T: rotation of catalytic and regulatory subunits
• binding of CTP to regulatory subunit stabilizes T form
• in absence of any S or regulator, T is favored (200:1)
• equilibrium constant explains the rate of converting R to T is 200x faster than T to R
• T favored by CTP binding; R favored by S binding
• Lys84 and Ser80 interactions shift into place in T-R transition and create high-affinity BS for S

Question 10

ATCase shows sigmoidal kinetics

Answer 10

• non MM kinetics
• active site cooperates (cooperativity = binding of one ligand at one site affects binding at other sites)
• sigmoidal cure is linear combination of two different MM curves but with different Km values
• R curve; v. tight binding, so at low [S], v. quickly reaches Vmax
• T curve: goes to same Vmax level but at much higher [S]

Question 11

allosteric regulation T-R equilibrium (sigmoidal curve)

Answer 11

• in prescence of feedback inhibition from heterotropic allosteric inhibitor CTP, at same [S] enzyme rate is reduced and shifts curve to right
• ATP can signal that there’s energy available for RNA synthesis so shift to left and favor R and enzyme becomes more active so at same [S] stimulates faster conversion