Evidence of a low-barrier hydrogen bond in the tryptophan synthase catalytic mechanism

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(Aex(1)), 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-H-2-D,L-Ser is substituted for alpha-H-1-D,L-Ser, the position of equilibrium is shifted in favor of E(Aex(1)). On a much slower time scale, the H-2 sample undergoes the exchange of enzyme bound H-2 for the H-1 of solvent water and is converted to a distribution of E(Aex(1)) and E(A-A) identical to that obtained with the H-1 sample. This slow exchange indicates that the proton abstracted from the alpha-carbon of E(Aex(1)) 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(Aex(1)) and E(A-A) of K-H/K-D = 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(Aex(1)). 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(Aex(1)), 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.