Secondary kH/kT kinetic isotope effects in H2O and kH/kT or kD/kT isotope effects in D2O have been measured for the triosephosphate isomerase-catalyzed conversion of dihydroxyacetone 3-phosphate (DHAP) to D-glyceraldehyde 3-phosphate. The proton transfer steps are made rate-limiting using [1(R)-2H]-labeled substrate in D2O to slow the chemical steps, relative to product release. After a small correction for the beta-equilibrium isotope effect for dehydration of DHAP, the H/T kinetic isotope effect kH/kT = 1.27 +/- 0.03 for [1(R)-2H,(S)-3H]-labeled substrate in D2O is subtantially larger than the equilibrium isotope effect for enolization of DHAP, KH/KT = 1.12. The H/T isotope effect is related to the D/T isotope effect with a Swain-Schaad exponent y = 4.4 +/- 1.3. These results are consistent with coupled motion of the C-1 primary and secondary hydrogens of DHAP and tunneling. Large secondary kinetic isotope effects are a general feature of enzymatic enolization reactions while nonenzymatic enolization reactions show secondary kinetic isotope effects that are substantially smaller than equilibrium effects [Alston, W. A., II, Haley, K., Kanski, R., Murray, C.J., & Pranata, J. (1996) J. Am. Chem Soc., 118, 6562-6569]. Possible origins for these differences in transition state structure are discussed.

This review compiles the combined chemical and enzymatic synthesis of aromatic l-amino acids (l-phenylalanine, l-tyrosine, l-DOPA, l-tryptophan, and their derivatives and precursors) specifically labeled with carbon and hydrogen isotopes, which were elaborated in our research group by the past 20 years. These compounds could be then employed to characterize the mechanisms of enzymatic reactions via kinetic and solvent isotope effects methods.

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Nine isotopomers of tryptamine and its halogen derivatives, labeled with deuterium, tritium in side chain, i.e., [(1R)-2H]-, [(1R)-3H]-, 5-F-[(1R)-2H]-, 5-F-[(1R)-3H]-, 5-Br-[(1R)-2H]-, double labeled [(1R)-2H/3H]-, 5-F-[(1R)-2H/3H]-, and ring labeled [4-2H]-, and [5-2H]-tryptamine, were obtained by enzymatic decarboxylation of l-Trp and its appropriate derivatives in deuteriated or tritiated media, respectively. Intermediates: [5′-2H]-l-Trp used for further decarboxylation was synthesized by enzymatic coupling of [5-2H]-indole with S-methyl-l-cysteine, and [4′-2H]-l-Trp was obtained by isotope exchange 1H/2H of the authentic l-Trp dissolved in heavy water induced by UV-irradiation. Doubly labeled [(1R)-2H/3H]- and 5-F-[(1R)-2H/3H]-tryptamine were obtain by decarboxylation of l-Trp or [5′-F]-l-Trp carried out in 2H3HO incubation medium.