Catalytic mechanism of the tryptophan synthase alpha(2)beta(2) complex - Effects of pH, isotopic substitution, and allosteric ligands

The mechanism of the tryptophan synthase alpha(2)beta(2) complex from Salmo nella typhimurium is explored by determining the effects of pH, of temperat ure, and of isotopic substitution on the pyridoxal phosphate-dependent reac tion of L-serine with indole to form L-tryptophan, The pH dependence of the kinetic parameters indicates that three ionizing groups are involved in su bstrate binding and catalysis with pK(a)1 = 6.5, pK(a)2 = 7.3, and pK(a)3 = 8.2-9. A significant primary isotope effect (similar to 3.5) on V and V/K is observed at low pH (pH 7), but not at high pH (pH 9), indicating that th e base that accepts the alpha-proton (beta Lys-87) is protonated at low pH, slowing the abstraction of the alpha-proton and making this step at least partially rate- limiting. pK(a)2 is assigned to beta Lys-87 on the basis of the kinetic isotope effect results and of the observation that the competi tive inhibitors glycine and oxindolyl-L-alanine display single pK(i) values of 7.3. The residue with this pK(a) (beta Lys-87) must be unprotonated for binding glycine or oxindolyl-L-alanine and, by inference, L-serine. Invest igations of the temperature dependence of the pK(a) values support the assi gnment of pK(a)2 to beta Lys-87 and suggest that the ionizing residue with pK(a)1 could be a carboxylate, possibly beta Asp-305, and that the residue associated with a conformational change at pK(a)3 may be beta Lys-167. The occurrence of a closed to open conformational conversion at high pH is supp orted by investigations of the effects of pH on reaction specificity and on the equilibrium distribution of enzyme-substrate intermediates.