common rate-determining step. Which of the possible hydrogen transfers is rate determining can be shown by measuring the kinetic effects of substituting each hydrogen by deuterium, its stable isotope. The rate will change when the critical hydrogen is replaced, due to a primary kinetic isotope effect, which occurs because bonds to deuterium are harder to break than bonds to hydrogen. It is also possible to measure similar effects with other isotope substitutions, such as 13C/12C and 18O/16O, but these effects are more subtle. Isotopes can also be used to reveal the fate of various parts of the substrate molecules in the final products. For example, it is sometimes difficult to discern the origin of an oxygen atom in the final product; since it may have come from water or from part of the substrate. This may be determined by systematically substituting oxygen's stable isotope 18O into the various molecules that participate in the reaction and checking for the isotope in the product. The chemical mechanism can also be elucidated by examining the kinetics and isotope effects under different pH conditions, by altering the metal ions or other bound cofactors, by site-directed mutagenesis of conserved amino acid residues, or by studying the behaviour of the enzyme in the presence of analogues of the substrate(s). Enzyme inhibition and activation Enzyme inhibitors are molecules that reduce or abolish enzyme activity, while enzyme activators are molecules that increase the catalytic rate of enzymes. These interactions can be either reversible (i.e., removal of the inhibitor restores enzyme activity) or irreversible (i.e., the inhibitor permanently inactivates the enzyme). Reversible inhibitors Traditionally reversible enzyme inhibitors have been classified as competitive, uncompetitive, or non-competitive, according to their effects on KM and Vmax. These different effects result from the inhibitor binding to the enzyme E, to the enzyme–substrate complex ES, or to both, respectively. The division of these classes arises from a problem in their derivation and results in the need to use two different binding constants for one binding event. The binding of an inhibitor and its effect on the enzymatic activity are two distinctly different things, another problem the traditional equations fail to acknowledge. In noncompetitive inhibition the binding of the inhibitor results in 100% inhibition of the enzyme only, and fails to consider the possibility of anything in between. In noncompetitive inhibition, the inhibitor will bind to an enzyme at its allosteric site; therefore, the binding