Evidence of a Low-Barrier Hydrogen Bond in the Tryptophan Synthase Catalytic Mechanism

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Abstract
In the absence of other substrates, l-Ser reacts rapidly with the tryptophan synthase α2β2 bienzyme from Salmonella typhimurium at pH 7.8 and 25 °C to give an equilibrating mixture of species dominated by comparable amounts of the l-Ser external aldimine Schiff base, E(Aex1), and the α-aminoacrylate Schiff base, E(A-A). The d-isomer of Ser is unreactive toward α2β2, 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 α-2H-d,l-Ser is substituted for α-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 α-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 α-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 α-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.