binding and enzymes

Question 1

what is the difference between the curves produced when oxygen binds to myoglobin vs haemoglobin?

Answer 1

haemoglobin exhibits a sigmoidal curve rather than hyperbola, due to co-operative binding

Question 2

define KD

Answer 2

the equilibrium dissociation constant, the concentration of ligand for half of maximum binding

Question 3

what is the difference in KD between a tight complex and a weak complex?

Answer 3

a tight complex has a very small value for KD, whereas a weak complex has a large value

Question 4

what determines the tightness of a complex?

Answer 4

deltaG; a tight complex will have a large value for deltaG, iy is very stable and the energy change is difficult to overcome

Question 5

why is KD useful?

Answer 5

its a measurement of affinity, affinity reflects function

Question 6

explain how selectin-carbohydrate interactions are an example of a weak complex

Answer 6

selectin-mediated adhesion to leukocyte sialylyl-lewis is weak, this permits leukocytes to roll along the vascular endothelial surface. transient interactions slow the white blood cell down

Question 7

how does equilibrium dialysis work?

Answer 7

• the membrane is permeable only to L
• over time, some ligand binds to protein and can no longer pass through the membrane
• concentration of free ligand decreases

Question 8

what is the relevance of equilibrium dialysis?

Answer 8

measuring bound or free protein at equilibrium allows you to determine KD

Question 9

what does isothermal titration calorimetry measure?

Answer 9

heat released (in an exothermic reaction) or absorbed (in an endothermic reaction) upon binding

Question 10

what does analytical ultracentrifugation measure?

Answer 10

the distribution of molecular weights of molecules. a complex will sediment faster than the individual components

Question 11

what are the limitations of equilibrium methods?

Answer 11

[L] or [P] and [PL] must be accurately measurable

the chosen technique must be sensitive in the range of the dissociation constant

Question 12

what is the rate of reaction determined by?

Answer 12

the magnitude of its activation energy

Question 13

what is association rate?

Answer 13

k1[P][L]

a measure of the number of productive collisions per unit of time

Question 14

what is rate of association driven by?

Answer 14

limited by rate of diffusion, however, can be faster than rate of diffusion due to local electrostatic interactions

Question 15

what is dissociation rate?

Answer 15

K-1[PL]

measure of the rate of separation of protein and ligand

Question 16

what is surface plasmon resonance?

Answer 16

a technique which detects changes in the refractive index in the immediate vicinity of the surface layer of a sensor chip. refractive index is dependent on the mass of material on the surface of the chip

Question 17

what is essential for a steady-state reaction?

Answer 17

substrate in excess

Question 18

in a steady-state reaction, what is the order with respect to the substrate?

Answer 18

initially first order, as increased further, reaction behaves as if zero order

Question 19

what occurs in the ‘pre-steady state’ phase?

Answer 19

enzyme binds to its substrate

Question 20

when might dissociation constant be equal to the Michaelis constant?

Answer 20

when K2 is very small compared to K-1