To properly address this problem, you should use the correct rate equation for a 2-substrate enzyme with substrate inhibition. You will need to know about the kinetic mechanism in order to select the right rate equation. There are several possibilities for steady-state bisubstrate kinetics, including but not limited to Rapid Equilbrium Random, Ordered, and Ping Pong. To find these equations, consult an advanced text on steady-state enzyme kinetics, such as the one by I. Segel. Then, you can model what should happen when the substrate concentrations are changed. To do this properly, you will also need to know the values of the kinetic constants (Kms, Vmax, and Kia if appropriate). You should measure these by varying the concentrations of both substrates in a matrix (for example, 6 concentrations of each, for a total of 36 combinations), then fit the full set of data to the rate equation using nonlinear regression software.

The kinetics of enzymes catalysing reactions with two or more substrates can be complex and somehow degenerate (not everything can be learned from simple steady-state measurements). In order to unravel the complexities behind the kinetic mechanism of your enzyme you need to do full initial-velocity patterns (a matrix of velocity as a function of changes in both substrates from low to nearly saturating conditions), and depending on the results you may have to do some additional tests to discriminate between alternatives.

There are many old papers of enzymes with two substrates and suffering substrate inhibition, and the methods used, despite the time elapsed are still valid today. A couple of examples:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1198559/?page=1

http://www.sciencedirect.com/science/article/pii/S0167483897000599

In the second paper I am a coauthor,  and I have shared with you in the RG system privately.

Best wishes,

Rogelio

Dear all,

Thank you for the helpful replies. I have measured the velocity with varying concentrations of both substrates in a matrix. The 1/v versus 1/[A] plots are linear at all B (inhibiting substrate) concentrations and the slopes of the plots increase with increasing [B]. Could this suggest the formation of a dead-end EB complex? The data fit well to such a velocity equation: v=Vmax[A][B]/(KiaKmb(1+[B]/Ki)+Kma[B](1+[B]/Ki)+Kmb[A]+[A][B]).

Best,

Kasper

Substrate inhibition by B in a bisubstrate reaction should result in curved reciprocal plots of 1/rate v. 1/[B].

In normal space (rate v.[B]), you should see a peak rate followed by a decreased rate as [B] increases.

Rather than using reciprocal plots, I recommend using nonlinear regression to fit the entirety of the data to the rate equation in order to obtain best-fit values of the kinetic constants.