My enzyme has 3 substrates. I want to determine Kd of each substrates using isothermal titration calorimetry. However, the first substrate does not "bind" with the enzyme unless the second substrate is present (and vice-versa). Only when both substrates are titrated will I get a signal using ITC. Are the results valid?
For your 3-substrate enzyme, whether or not you see heat of reaction in ITC when only 2 substrates are added depends upon the reaction mechanism.
For example, in the sequential ternary complex mechanism below, you need all 3 substrates to bind before any reaction occurs.
E + A EA
EA + B EAB
EAB + C EABC EPQR
You can add B to a mixture of E and A to measure the heat of binding of B, but you can't add C to a mixture of E+A+B.
In contrast, in the Ping-Pong mechanism example below, a partial reaction occurs when you add B to EA, so you can't measure the heat of binding when you add B to E+A, or C to E+A+B either.
E + A EA
EA + B EAB EQ + P
EQ + C ER
In a rapid equilibrium random ternary complex mechanism in which the substrates can bind in any order, you could add any one substrate, or any 2 of the 3 substrates without getting a reaction.
In some cases, you could add the 3 substrates but prevent the reaction from occurring by leaving out a cosubstrate like Mg2+. The downside is that the substrate affinities may be different with and without it.
Some of the Kds can be determined using steady-state kinetics of the forward reaction if you know the rate equation. These experiments are also useful for giving Kms.
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