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 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.
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Laboratory of Biochemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0830, USA.
Wilson Miles
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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*
D007211:Indoles; D008024:Ligands; D000078183:Oxindoles; C044832:oxindolylalanine; D012694:Serine; D011732:Pyridoxal Phosphate; C030374:indole; D003903:Deuterium; D014367:Tryptophan Synthase; D000409:Alanine; D005998:Glycine