The Mechanism of Binding of L‐Serine to Tryptophan Synthase from Escherichia coli

Publisher Website
Google Scholar
Abstract
The mechanism of binding of L-serine to tryptophan synthase, which is the initial phase of the catalytic mechanism, was studied by steady-state and stopped-flow kinetic techniques. The dependence of 3 separable rate processes on the concentration of L-serine is compatible with 4 different enzyme-substrate complexes, one of which lies on a branch in the pathway. By use of L-serine deuterated at the .alpha. C, 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 2 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. C 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 the rate of deprotonation and .beta.-elimination, shifting the rate-limiting step to the release of L-tryptophan.