title: Catalytic mechanism of the tryptophan synthase alpha(2)beta(2) complex. Effects of pH, isotopic substitution, and allosteric ligands.
abstract: The mechanism of the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium is explored by determining the effects of pH, of temperature, and of isotopic substitution on the pyridoxal phosphate-dependent reaction of L-serine with indole to form L-tryptophan. The pH dependence of the kinetic parameters indicates that three ionizing groups are involved in substrate 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 (approximately 3.5) on V and V/K is observed at low pH (pH 7), but not at high pH (pH 9), indicating that the base that accepts the alpha-proton (betaLys-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 betaLys-87 on the basis of the kinetic isotope effect results and of the observation that the competitive inhibitors glycine and oxindolyl-L-alanine display single pK(i) values of 7.3. The residue with this pK(a) (betaLys-87) must be unprotonated for binding glycine or oxindolyl-L-alanine, and, by inference, L-serine. Investigations of the temperature dependence of the pK(a) values support the assignment of pK(a)2 to betaLys-87 and suggest that the ionizing residue with pK(a)1 could be a carboxylate, possibly betaAsp-305, and that the residue associated with a conformational change at pK(a)3 may be betaLys-167. The occurrence of a closed to open conformational conversion at high pH is supported by investigations of the effects of pH on reaction specificity and on the equilibrium distribution of enzyme-substrate intermediates.
authors.affiliation: Laboratory of Biochemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0830, USA.
authors.forename: H S
authors.initials: HS
authors.lastname: Ro
authors.forename: E
authors.initials: E
authors.lastname: Wilson Miles
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000494:pharmacology; D000494:Allosteric Regulation; D020134:Catalytic Domain; D003903:Deuterium; D005998:Glycine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000378:metabolism; D008024:Ligands; D008956:Models, Chemical; D000078183:Oxindoles; D011732:Pyridoxal Phosphate; D012486:Salmonella typhimurium / Q000201:enzymology*; D012694:Serine / Q000378:metabolism; D014367:Tryptophan Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D007211:Indoles; D008024:Ligands; D000078183:Oxindoles; C044832:oxindolylalanine; D012694:Serine; D011732:Pyridoxal Phosphate; C030374:indole; D003903:Deuterium; D014367:Tryptophan Synthase; D000409:Alanine; D005998:Glycine