Energetics of proline racemase: double fractionation experiment, a test for concertedness and for transition-state dominance.

To test whether a reaction involving the making and/or breaking of two bonds at two sites is concerted (and proceeds through a single transition state) or is stepwise (and involves a reaction intermediate in which only one bond has been made or broken), we have measured the isotopic fractionation at one site as a function of isotopic substitution at the other site. In the case of proline racemase, the discrimination against solvent deuterium in the product when the reaction is run in mixed H2O-D2O is measured for the reaction both of [2-1H]proline and of [2-2H]proline. The isotopic fractionation at the solvent site may in principle be smaller, the same, or larger, when the 2H-labeled substrate is used rather than the 1H substrate, and--depending upon the nature of the catalyzing groups--this information indicates whether the reaction is stepwise, or concerted, or whether an isotopically insensitive transition state is partially rate determining. Experimentally, we have found that the discrimination against solvent deuterium in the product L-proline is the same, whether D-[2-1H]proline or D-[2-2H]proline is the substrate. This result requires that the substrate and product "on-off" steps are faster than the racemization step and that the racemization reaction proceeds either in a concerted manner or in a stepwise fashion involving enzyme catalytic groups (e.g., thiols) having ground-state fractionation factors around 0.5.