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 α 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 β2 subunit than to the α2β2 complex of tryptophan synthase and retains its α carbon proton. For the β2 subunit, the rate‐determining step of tryptophan synthesis is deprotonation of bound L‐serine. The effect of bound α subunit is to increase both the rate of deprotonation and β‐elimination, shifting the rate‐limiting step to the release of L‐tryptophan.
|Number of pages||10|
|Journal||European Journal of Biochemistry|
|State||Published - Jan 1983|
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