A proton-magnetic-resonance study of hydrogen-exchange reactions of yeast tryptophan synthase.
1H n.m.r. was used to observe tryptophan formation from indole and L-serine, proton exchange at C-2 of L-tryptophan, and proton exchange at C-2 of L-serine, catalysed by yeast tryptophan synthase in the presence of 2H2O. Tryptophan synthesis took place with compulsory replacement of C-2 hydrogen by solvent hydrogen. The exponential decay rate (kobs) of the serine exchange reaction was insensitive to serine concentration in the range 2-20mM and was used to calculate kcat./Km values. However, kobs. was very sensitive to pH* values in the range 6.5-8.5 and the data require that the free enzyme is active in the base form resulting from two inseparable ionizations of pKa 7.3, and inactive after a third ionization controlled by a pKa of 7.5. Initial rates measured by u.v. absorbance and colorimetric procedures were used to calculate kinetic parameters of the tryptophan synthesis reaction. From pH 6.5 to 7, kcat./Km values for L-serine in the tryptophan synthesis and hydrogen exchange reactions were indistinguishable and increased rapidly under the control of two acid-base groups of pKa 6.7 and 7.2. Above pH 7, this equivalence breaks down because the exchange reaction alone is responsive to the third pKa value of the free enzyme. The pH dependence of the catalytic constant for tryptophan synthesis was qualitatively similar to that of the kobs. for serine exchange. A mechanism to explain the results is contrasted with recent proposals for the Escherichia coli system.
Bailey
C J
CJ
Department of Biochemistry, Trinity College, Dublin, Ireland.
Malthouse
J P
JP
D006859:Hydrogen; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008433:Mathematics; D011865:Radioisotope Dilution Technique; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012694:Serine / Q000378:metabolism; D014316:Tritium; D014364:Tryptophan / Q000302:isolation & purification; D014367:Tryptophan Synthase / Q000378:metabolism*
D014316:Tritium; D012694:Serine; D006859:Hydrogen; D014364:Tryptophan; D014367:Tryptophan Synthase