Solvent Kinetic Isotope Effects in Enzyme Reactions See Also Section

6 Solvent Kinetic Isotope Effects in Enzyme Reactions (See Also Section 11.4) 

As a practical matter of cost, studies on solvent isotope effects are usually limited to D/H substituted solvents, although recently a few lsO solvent effects have been measured. Interpretation of enzymatic solvent isotope effects is even more complicated than it is when the isotopic probe is incorporated in the substrate(s). This is because enzyme proteins have many exchangeable protons and, also, this is frequently true for reactants (substrates). Thus the observed isotope effect is the collective result of many different isotopic substitutions, each of which may influence 

A similar analysis applies to CBS mutated tetrachlorohydroquinone dehalo-genase but normal rather than inverse solvent isotope effects are involved. This 

An S Ar (nucleophilic substitution at aromatic carbon atom) mechanism has been proposed for these reactions. Both nonenzymatic and enzymatic reactions that proceed via this mechanism typically exhibit inverse solvent kinetic isotope effects. This observation is in agreement with the example above since the thiolate form of glutathione plays the role of the nucleophile role in dehalogenation reactions. Thus values of solvent kinetic isotope effects obtained for the C13S mutant, which catalyzes only the initial steps of these reactions, do not agree with this mechanism. Rather, the observed normal solvent isotope effect supports a mechanism in which step(s) that have either no solvent kinetic isotope effect at all, or an inverse effect, and which occur after the elimination step, are kinetically significant and diminish the observed solvent kinetic isotope effect. 

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