Enzyme Mechanism Problems - (Jul/02/2014 )

Hi

I am posting here for the first time. I am trying to determine the mechanism of an enzyme catalyzed reaction. It has two substrates. I found that the enzyme is inhibited by one of its substrate and follows a compulsory order ternary complex mechanism. 

I am quite confident about my data but my supervisor thinks I should do product inhibition studies in order to confirm this mechanism. I have no experience with this and I am not sure if this will be a correct way to do it because substrate inhibition will make it more complicated.

Any suggestions will be very helpful and I thank you in advance. 

have you determined the Km(s) of the substrate(s) for the enzyme?

if so, then, using a fixed substrate concentration equal to the Km, use a range of product concentrations to determine if you are seeing inhibition and, if so, determine the Ki.

Thank you very much I appreciate it.

Yes I have determined the Km(s) of the substrates (I atleast have approximate values).

But I am confused here by what does "fixed" concentration mean? because as the reaction proceeds the amount of substrate will reduce. 

For example:- If I have 5mM of substrate in the reaction mixture (200microL) and I am adding 1microL of 10mM ATP every 1 minute and measuring rate. Which of this will be fixed and which will be variable?

this may seem counter-intuitive to you but the 5mM substrate is considered to be "fixed". if you continue adding atp then it is variable (in my experience, this is not a routine procedure).

this type of assay is to determine "steady-state" kinetics, you add all components, incubate for a period of time, then evaluate the result.

Your answer helped me understand something. Thank you. :)

I am still not sure how to get product inhibition done when the substrate inhibits the reaction at higher concentrations. Or is it even worth doing product inhibition experiments?

it could be the product causing the inhibition. performing the product inhibition studies will help determine this.

also, it may not be the substrate causing the inhibition but its effect on the environment (pH, ionic strength, etc) that may be causing inhibition.

Thanks. I do not think its product inhibition as the product formed during the experiment is very low as compared to the substrate. But it could be other environmental factors causing inhibition.