<Li> Non-competitive inhibitors have identical affinities for E and ES (K = K'). Non-competitive inhibition does not change K (i.e., it does not affect substrate binding) but decreases V (i.e., inhibitor binding hampers catalysis). </Li> <Li> Mixed - type inhibitors bind to both E and ES, but their affinities for these two forms of the enzyme are different (K ≠ K'). Thus, mixed - type inhibitors interfere with substrate binding (increase K) and hamper catalysis in the ES complex (decrease V). </Li> <P> When an enzyme has multiple substrates, inhibitors can show different types of inhibition depending on which substrate is considered . This results from the active site containing two different binding sites within the active site, one for each substrate . For example, an inhibitor might compete with substrate A for the first binding site, but be a non-competitive inhibitor with respect to substrate B in the second binding site . </P> <P> As noted above, an enzyme inhibitor is characterised by its two dissociation constants, K and K', to the enzyme and to the enzyme - substrate complex, respectively . The enzyme - inhibitor constant K can be measured directly by various methods; one extremely accurate method is isothermal titration calorimetry, in which the inhibitor is titrated into a solution of enzyme and the heat released or absorbed is measured . However, the other dissociation constant K' is difficult to measure directly, since the enzyme - substrate complex is short - lived and undergoing a chemical reaction to form the product . Hence, K' is usually measured indirectly, by observing the enzyme activity under various substrate and inhibitor concentrations, and fitting the data to a modified Michaelis--Menten equation </P>

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