In the absence of other substrates, L-Ser reacts rapidly with the tryptophan synthase alpha 2 beta 2 bienzyme from Salmonella typhimurium at pH 7.8 and 25 degrees C to give an equilibrating mixture of species dominated by comparable amounts of the L-Ser external aldimine Schiff base, E(Aex1), and the alpha-aminoacrylate Schiff base, E(A-A). The D-isomer of Ser is unreactive toward alpha 2 beta 2, and therefore, D,L-Ser can be used in place of L-Ser for investigations of catalytic mechanism. Due to the equilibrium isotope effect, when alpha-2H-D,L-Ser is substituted for alpha-1H-D,L-Ser, the position of equilibrium is shifted in favor of E(Aex1). On a much slower time scale, the 2H sample undergoes the exchange of enzyme bound 2H for the 1H of solvent water and is converted to a distribution of E(Aex1) and E(A-A) identical to that obtained with the 1H sample. This slow exchange indicates that the proton abstracted from the alpha-carbon of E(Aex1) is sequestered within a solvent-excluded site in E(A-A). Analysis of the UV/vis spectra gave an isotope effect on the equilibrium distribution of E(Aex1) and E(A-A) of KH/KD = 1.80 +/- 0.18. This large equilibrium isotope effect is the consequence of an unusual isotope fractionation factor of 0.62 for the residue which functions as the base to deprotonate and protonate the alpha-carbon proton in E(Aex1). A fractionation factor of 0.62 qualifies as evidence for the involvement of a low-barrier H-bond (LBHB) in this equilibration. Since this effect arises from abstraction of the alpha-proton from E(Aex1), the LBHB must be associated with the E(A-A) species. In contrast to weak H-bonds with energies of 3-12 kcal/mol, LBHBs are proposed to exhibit energies in the 12-24 kcal/mol range [Frey, P.A., Whitt, S.A., & Tobin, J. B. (1994) Science 264, 1927-1930]. Possible roles for this LBHB both in the chemical mechanism and in the stabilization of the closed conformation of E(A-A) are discussed.