If you are setting up an enzyme assay to identify inhibitors and/or measure their potencies, you should begin by measuring the Km of the substrate. The concentration of the substrate relative to its Km can have a big effect on the measured potency of the inhibitor, if the inhibitor is either a competitive or uncompetitive inhibitor. Please read the book Evaluation of Enzyme Inhibitors in Drug Discovery by Robert A. Copeland for more details.