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The small magnitudes of some kinetic isotope effects (KIEs), including those associated with 13C, necessitate a highly precise experimental approach involving the competition of light and heavy substrates. Provided the reaction is first order in the labeled substrate, the product isotopologue ratio converges to the initial reactant isotopologue ratio at completion, but the same is not true for dimerization reactions simply because the product diverges into four distinct isotopologues. The relative populations of these dimers deviate from the statistical distribution under the influence of a KIE. Accordingly, the current study aims to demonstrate this concept by analyzing the relative 13C placement in D-alanine-D-alanine at reaction completion for the dimerization of D-[1-13C]alanine catalyzed by Mycobacterium tuberculosis D-alanine:D-alanine ligase (Ddl). Using 13C NMR spectroscopy and Fourier-transform ion cyclotron mass spectrometry, the relative distributions of the four dimer isotopologues were determined. The ratio of the mono-labeled dimers with 13C at the C-terminus to that with 13C at the N-terminus yielded a relative KIE of 1.011 ± 0.004 for the acyl carbon. This result suggests that the rate-limiting step of the Ddl-catalyzed reaction involves peptide bond formation—either nucleophilic attack by the amino group or collapse of the resulting tetrahedral intermediate. This method of analysis, to the best of our knowledge, is the first of its kind for obtaining competitive KIEs in enzyme-catalyzed dimerization reactions. 

Solvent isotope effects have long been used as a mechanistic tool for determining enzyme mechanisms. Most commonly, macroscopic rate constants such as kcat and kcat/Km are found to decrease when the reaction is performed in D2O for a variety of reasons including the transfer of protons. Under certain circumstances, these constants are found to increase, in what is termed an inverse solvent kinetic isotope effect (SKIE), which can be a diagnostic mechanistic feature. Generally, these phenomena can be attributed to an inverse solvent equilibrium isotope effect on a rapid equilibrium preceding the rate-limiting step(s). This review surveys inverse SKIEs in enzyme-catalyzed reactions by assessing their underlying origins in common mechanistic themes. Case studies for each category are presented, and the mechanistic implications are put into context. It is hoped that readers may find the illustrative examples valuable in planning and interpreting solvent isotope effect experiments.