Question

How to measure km and vmax of the enzyme?

Answer 1

How to calculate the km and Vmax values of an enzyme when I have substrate/product inhibition?

Hello,

Could anyone please help me in calculating the Km and Vmax values of an enzyme (I am working on dihydrofolate reductase DHFR) when I have substrate/product inhibition?

I see the inhibition on (specific activity nmol/min/mg to substrate concentration µM) curve, with substrate concentrations near the theoretical Vmax. (I mean the calculated Vmax by ignoring the inhibition).

(I attached a link of the curve)

- More details:

the Km of my enzyme is about 1µM. (in case I ignored the inhibition).

The specific activity with 5µM substrate is 2µM/min/mg. (in my case this is Vmax when I ignore the inhibition).

The specific activity with 10µ substrate is 1,7µM/min/mg. (it is lower here, that means here we have inhibition).

The specific activity with 20µM substrate is 1,5µM/min/mg. (again inhibition).

I don't know what is the right method to calculate the Vmax and Km for that enzyme and substrate with that inhibition phenomenon,


waiting kindly for your guiding.

Regards.

Answer 2

It is important to have as thorough knowledge as is possible of the performance characteristics of enzymes, if they are to be used most efficiently. The kinetic parameters Vmax, Km and kcat/Km should, therefore, be determined. There are two approaches to this problem using either the reaction progress curve (integral method) or the initial rates of reaction (differential method). Use of either method depends on prior knowledge of the mechanism for the reaction and, at least approximately, the optimum conditions for the reaction. If the mechanism is known and complex then the data must be reconciled to the appropriate model (hypothesis), usually by use of a computer-aided analysis involving a weighted least-squares fit.