Aqueous Solvent Equilibrium and Kinetic Isotope Effects

4 Aqueous Solvent Equilibrium and Kinetic Isotope Effects 

It is reasonable to expect that isotopic substitution on solvent molecules will affect both equilibrium and rate constants. This is especially true for reactions in aqueous media, many of which are acid or base catalyzed and therefore sensitive to pH or pD. Furthermore H/D aqueous solvent isotope effects often display significant nonlinearity when plotted against isotope fraction of the solvent. The analysis of this effect can yield mechanistic information. The study of aqueous solvent isotope effects is particularly important in enzyme chemistry because enzyme reactions universally occur in aqueous media and are generally pH sensitive. 

The isotope effect on the acid dissociation constants for H20 and D20 has been carefully measured by many workers, most notably Paabo and Bates working at the US Bureau of Standards. Comparing the reactions 2 H20 = H30+ + OH- and 2 D20 = D30+ + OD they found 

In other words the acid dissociation constant of ordinary water, Ka(H20) = 1 x 10-14, is about ten times larger than that of heavy water, Ka(D20) = 1.10 x 1CT15. Thus the pH of ordinary water is pH = - log((H30+)) = - log[(l x 10-14)1/2] = 7.00 while pD for D20 is 7.48. This is a significantly large isotope effect and has important consequences. 

Many rate constants in aqueous solutions are pH or pD sensitive. In particular, enzyme catalyzed reactions often show maxima in plots of pH(pD) vs. rate. The example in Fig. 11.5 is constructed for a reaction with a true isotope effect, kH/kD = 2, and with maxima in the pH(pD)/rate dependences as shown by the bell shaped curves. These behaviors are typical for enzyme catalyzed reactions. When the isotope effect is obtained (incorrectly) by comparing rates at equal pH and pD, the values plotted along the steep dashed curve result. If, however, the rate constants at corresponding pH and pD (pD = pH + 0.5) are employed, a constant and correct value is obtained, kH/kD = 2. Thus for accurate measurements of the isotope effects one must control pH and pD at appropriate values (pD = pH + 0.5 in our example) using a series of buffers. In proton inventory experiments (see below) buffers should be employed to insure equivalent acidities across the entire range of solvent isotope concentration (0 xD 1), xD is the atom fraction of deuterium [D]/([H] + [D]). 

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