The mechanism of binding of L-serine to tryptophan synthase from Escherichia coli.

The mechanism of binding of L-serine to tryptophan synthase, which is the initial phase of the catalytic mechanism, has been studied by steady-state and stopped-flow kinetic techniques. The dependence of three separable rate processes on the concentration of L-serine is compatible with four different enzyme-substrate complexes, one of which lies on a branch in the pathway. By use of L-serine deuterated at the alpha carbon, it is possible to assign the deprotonation of the external aldimine of L-serine with pyridoxal 5'-phosphate to the most rapid observable binding step. Measurements at two pH values show that the rate-determining step in the synthesis of L-tryptophan changes from release of L-tryptophan at the optimal pH of 7.6 to the binding of L-serine at pH 6.5. Measurements at pH 7.6 in the presence of the substrate analogue indolepropanol phosphate show that the stronger binding of L-serine is probably due to stabilization of the catalytically competent enzyme--L-serine complex. At pH 7.6 L-serine is bound far more slowly to the beta 2 subunit than to the alpha 2 beta 2 complex of tryptophan synthase and retains its alpha carbon proton. For the beta 2 subunit, the rate-determining step of tryptophan synthesis is deprotonation of bound L-serine. The effect of bound alpha subunit is to increase both the rate of deprotonation and beta-elimination, shifting the rate-limiting step to the release of L-tryptophan.