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

Abstract

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.

On the electronic nature of low-barrier hydrogen bonds in enzymatic reactions

“…This is in agreement with the very high 1 H chemical shifts measured by NMR for protons proposed to be involved in LBHB formation (5)(6)(7)(8)(9)(10)(11)(12). The emerging picture of LBHBs as having covalent bonds between charged atoms is thus in good agreement with this special spectroscopic characteristic of the LBHB systems.…”

“…It has been suggested that formation of ''short-strong'' hydrogen bonds (SSHB) or ''low-barrier'' hydrogen bonds (LBHB) can stabilize intermediates and͞or transition states of enzymatic reactions (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Originally, the involvement of LBHB in enzymatic reactions was suggested by Cleland (1) based on the observation of unusually low hydrogen fractionation factors for certain enzymes.…”

Structure and Function of the Tryptophan Synthase α2β2 Complex

“…The presence of the hydrogen bond between His 86 and Lys 87 in E-AA (19) is consistent with evidence from spectroscopic experiments that the abstracted ␣-proton of L-Ser is sequestered in a solvent-excluded site and possibly stored in a low barrier hydrogen bond (20 , and the phosphate of PLP. Indeed, the 31 P nuclear magnetic resonance chemical shift of the phosphate of the PLP, which is bound to the tryptophan synthase ␣ 2 ␤ 2 complex and ␤ 2 subunit from E. coli, is pH-independent from pH 6.5 to 8.6, whereas the PLP 31 P chemical shift of a model PLP derivative exhibits a pK a value of 6.23 (21).…”

Co-crystal/salt crystal structure disorder of trichloroacetic acid–N-methylurea complex with double system of homo- and heteronuclear O–H···O/N–H···O hydrogen bonds: X-ray investigation, ab initio and DFT studies

“…Many researches are devoted to very strong hydrogen bonds, because of their important role as a transition state in biochemical reactions and enzyme catalysis [6][7][8][9][10][11][12]. Their unusual stabilization energy makes them to be distinguished from ordinary hydrogen bonds.…”