Two Substrate Enzymology

Question 1

How can we distinguish between random sequential and ordered sequential mechanisms?

Answer 1

Protective effect of ligand binding on thermal denaturation:
Random = Both substrates alone protect, and more with A+B.

Ordered = Only 1st binding substrate protects alone, with second substrate (which has dependent binding) not protecting alone.
(Theorell chance is same - except A+B is less strong protection).

Equilibrium Dialysis:
Random = Each substrate alone will form ES complex (binary)…
Ordered = Only 1st binding substrate will form ES complex (binary) on its own…. second substrate, with dependent binding, won’t form ES complex when added on its own.

Kinetics in the presence of excess product:

Random = Each product, will show competitive inhibition for both substrates = 4 competitive profiles.
Ordered = 1st forming product will competitively inhibit the first binding substrate! Other 3 will be uncompetitive/mixed.

Question 2

How can we know mechanism from primary graphs alone?

Answer 2

Random sequential (Most likely independent and in rapid global equilibrium) = when BOTH intersects ON X-axis

Ping-Pong = parallel, non intersecting lines

Intersect above or below X-axis = Ordered sequential, with cooperativity or hindrance…. Or random dependent binding.

Question 3

How can we distinguish random sequential dependent and independent?

Answer 3

Random sequential dependent = random order of substrate binding, however binding of one substrate modifies affinity for binding the next.
Random independent = binding of the substrate does not change affinity for binding the next.

Equilbrium dialysis = can calculate dissociation constant, KL.
KL (conc.) = reflects affinity of enyme for ligand.

Equilbrium dialysis = if KL changes in presence of second substrate, then random dependent.

Question 4

How are constants KAB defined for the different mechanisms?

Answer 4

KAB = 0 for Ping-Pong = no ternary complex forms.

KAB = KMb*[A] = for Sequential ordered. = KAB is determined by binding of substrate A.

KAB = KMb[A] = KMa[B] = identical

Question 5

What is general rate equation of two substrate reaction?

Answer 5

Vo = Vmax * [A] * [B]
////
Kab + KmA[B] + KmB[A] + [A]*[B]

Question 6

What is global rapid equilbrium?

Answer 6

Steady State Mechanism.
Central isomerisation step of EAB -> EPQ is slowest step.
This central step controls the observed rate, with other steps in global rapid equilbrium.