Received January 31, 1996

Revised Manuscript Received March 21, 1996

Abstract:

In the absence of other substrates, L-Ser reacts rapidly with the tryptophan synthase ₂₂ 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 -aminoacrylate Schiff base, E(A-A). The D-isomer of Ser is unreactive toward ₂₂, 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 -²H-D,L-Ser is substituted for -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 -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 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 -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 -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.

Download the full text: PDF | HTML