Check out publications by Cleland et al to find out how to do experiments with two competing substrates and what are the rate equations. You can also try enzyme kinetics books such as those by Siegal. In these publications, you will find out how to set up your experiments.

First, there seems to be a contradiction (at least a semantic contradiction) in how you pose the problem. You ask: "... should I not measure the production of both B & Y cleavage products?" But then you also say, at the very end, that "B" and "Y" are actually identical. So measuring "production of both B & Y", as you say, really just means measuring production of "B" (which is the same as "Y").

Second, I agree with you completely that if, as you say, "many studies that claim that the competitive substrate X-Y is an inhibitor", that would be potentially confusing to many people. However, let's accept a definition of an "inhibitor" as a substance that suppresses the enzyme activity in comparison with an experiment conducted under otherwise exactly identical conditions except that the "inhibitor" is absent. Now under that particular definition the competing substrate is, in fact, an "inhibitor" is it not?

To repeat for clarity and use some actual numbers, let's say you do an assay with [E] = 1 nM and [A-B] = 10 µM. This produces the reaction rate v1. Now you do an assay with [E] = 1 nM, [A-B] = 10 µM and [X-Y] = 10 µM. This produces the reaction rate v2. If v2 is lower than v1, then X-Y is an "inhibitor" according to the operational definition we accepted above.

But now, what if you do an assay with [E] = 1 nM, [A-B] = 10 µM and [X-Y] = 0.1 µM, and now the reaction rate v3 is actually greater than v1? Then X-Y would be an "activator". Is this kind of a switch possible, at lest in principle? To find this out, you could use the free software package DynaFit and do a simulation under various conditions, changing the initial concentrations of A-B and X-Y. Here is where you can grab the program

http://www.biokin.com/dynafit/

And here is a DynaFit "script" I just used trying to understand the interesting problem that you posed (albeit somewhat confusingly):

______________________________________________

[task]

task = simulate

data = rates

approximation = rapid-equilibrium

[mechanism]

E + A-B E.A-B : Km(A-B) dissoc

E.A-B ---> E + A + B : kcat(A-B)

E + X-Y E.X-Y : Km(X-Y) dissoc

E.X-Y ---> E + X + B : kcat(X-Y)

[constants]

Km(A-B) = 10

Km(X-Y) = 20

kcat(A-B) = 1

kcat(X-Y) = 0.1

[concentrations]

E = 0.001

[responses]

B = 1000

[data]

variable X-Y

mesh logarithmic from 1 to 1000 step 2

directory ./project/compsub/data/sim-001

extension txt

plot logarithmic

file d01 | conc A-B = 0.1 | label [A-B] = 0.1

file d02 | conc A-B = 1 | label [A-B] = 1

file d03 | conc A-B = 10 | label [A-B] = 10

[output]

directory ./project/compsub/output/sim-001

[end]

______________________________________________

When I ran the script in DynaFit, it generated the output that is shown in the attached GIF image file. From this graph this you can see that at [A-B] = 0.1, the competing substrate is actually an "activator" of the enzyme; at [A-B] = 1 the competing substrate has no effect; and only at at [A-B] = 10 the competing substrate acts as an "inhibitor".

So, on the whole, I would agree with you completely that the terminology presumably used in "many studies", as you say, is very confusing.

Hope this helps,

-Petr

I would have mentioned Dynafit as well, as a first possible way to solve things but I did not know if it was available and/or easy enough to use. Thanks Petr for the explanation. I was not sure if I could give a correct response.

Thanks Marcia. I should let everyone know that DynaFit is always available at http://www.biokin.com/, since 1996. Currently there are 800+ journal publications that cited or used it. Last month the (British) Biochemical Society organized a one-day DynaFit workshop in London (http://www.biokin.com/courses/london2014). It seemed to have gone quite well so I am hoping we'll be back at the Charles Darwin house relatively soon.

http://www.biokin.com/dynafit/

Thank you Marcia and Petr for your comments. When I am ready to begin actual experiments the papers you have suggested Marcia, and the DynaFit program will be immensely useful. Petr: I have updated the question and also sent you more details in an email.