You could either confirm or exclude competitive inhibition by adding an excess of substrate that would keep the test going. From your graphs I don't think that your inhibitor is uncompetitive.

Good luck!

Yeah, this is part of the problem with Lineweaver-Burke plots.  They're very high in error.  Just based on what you're showing, the inhibitor is uncompetitive, since Vmax and Km both change, and binds to a binding site which has the substrate already bound.  But a good uncompetitive plot should have parallel lines, and yours clearly aren't parallel.  

I'd tend to suspect that you've actually got non-competitive inhibition (doesn't matter if substrate is bound) and that the lines SHOULD intersect at the x-axis.  But you can only do so much with LB plots.  Gisela is right, you'd probably be best off taking alternate approaches to determine the mechanism - and secondary validation is always a good idea anyways.  Adding a huge amount of substrate would eliminate competitive inhibition, but not other forms.  If you can engineer a direct binding readout, rather than a functional assay, you could validate uncompetitive inhibition by seeing if it binds the same or differently with or without substrate present.  Or you could try to get a crystal structure - there are a number of structures of your protein already in the pdb, so the crystallization conditions are probably reasonably well worked out.  Or you could try docking, and see where on the whole protein your molecule fits best, and if that is or is not the substrate binding site.

It seems to be a type of mixed inhibition altering Vmax and Km by different factors. With point of intersection lying below x axis causes decrease in Vmax and Km both. In another type of mixed inhibition point of intersection lies above x axis causing decrease in Vmax and increase in Km.  It is distinct from uncompetitive effect where these parameter are affected by same factor yielding parallel lines

It seems you have a bireactant system, whereby  the enzyme may  react with multiple substrates on account of its having multiple-sub-units  whereby  more than one is catalytic. Indeed glucronidase is a homotetramer and this may be create this BI BI kind of reaction mechanism.

Considering that the inhibitor is of relatively low potency, judging by the use of 100s of µM, and that it is probably the sort of aromatic compound usually found in small molecule screening libraries (the legend describes it as a pyrimidine derivative), your first concern before doing kinetic mode-of-inhibition studies should have been to convince yourself that the inhibition by this compound is specific and stoichiometric. It is a common occurrence when screening compound libraries to find non-specific inhibitors. Non-specific inhibition can take many forms. One well-described form is the existence of soluble compound aggregates that bind and inhibit the enzyme. Another is protein denaturation. Compound insolubility can cause inhibition. There can also be reactive contaminants. Finally, high compound concentrations can interfere with the assay.

An excellent way to test for specific inhibition is to measure the binding stoichiometry of the compound to the target. There are many methods for doing this. Isothermal titration calorimetry is an excellent one. Equilibrium dialysis is another. The stoichiometry should be 1 mole of inhibitor per mole of enzyme (if the enzyme is a monomer).

1-D ligand-observe NMR can be used to demonstrate inhibitor binding to the enzyme and test for competition by the substrate. 2-D protein-observe NMR experiments can be used to measure the binding affinity and test for specific binding.

There are several tests for non-specificity available. The inhibition should not change in potency when the enzyme concentration is increased (and the reaction time decreased accordingly to stay within initial rate conditions). The IC50 curve should have a Hill slope of 1. The IC50 should be lower than the solubility of the compound under assay conditions. The compound should not cause interference in the assay at the IC50 concentration. Adding 0.01% Triton X-100 detergent to the assay should not reduce the potency of the compound. A freshly prepared sample of the compound should be as potent as the original sample tested.

I do not understand your graph C.

Your plots (the ones above) as already indicated by Rekha and Yaman, show mixed-type (or general non-competitive in Cleeland's terms) with a Kic > Kiu, meaning the inhibitor binds better to the E.S complex than it does to the free E.

As pointed out by Williams, 1/v over 1/s plots have an statistical limitation, because the reciprocal of a small number is a big number, and this transformation makes small experimental errors to have a big influence on the uncertainty, and viceversa.

I also coincide with William in that you have not enough points to produce a good estimate of Kiu and Kic, but from your data Kic≈300 µM and Kiu≈150 µM.

You should use non-linear regression to fit the whole data matrix:

v1,1   [S]1    [I]1

v2,1   [S]2    [I]1

...

v1,2   [S]1   [I]2

v2,2   [S]2    [I]2

....

vm,n   [S]m   [I]n

To the mixed type inhibition equation:

v0=(VMAX·[S]/ (KM·(1+[I]/KIC)+[S]·(1+[I]/KIU))

If you have doubt about how to do so refer to this site:

http://statpages.info/nonlin.html

where you can perform the calculation (although you will not see a plot, you would have to plot the results with another program, for instance a spreadsheet with plotting capabilities).

Best wishes,

Rogelio

Not an expert on enzyme kinetics but for microbes the inhibitor concentrations are quite high and  contaminents, as one observer noted for enzymes, ed, can be quite problematic. One exasmple was commercial  14C amino acids  containing left over chemosynthetic reactants Don Button. 

At least two types of inhibition give that type pattern. They are diagrammed and discussed in Segal, Enzyme kinetics Wiley1993 957 pages. Both he and Cleland (See book on enzyme kinetics by Cook) were into examining mechanisms with inhibitors, particularly enzymes with multiple substrates. By the way I am working on a way that seems to work for determining the concentration of endogenous substrate via inhibition of radioactivity uptake with unlabeled substrate when background levels are lunknown. Would love to have someone review the MS.