Serine modulates substrate channeling in tryptophan synthase: A novel intersubunit triggering mechanism

Abstract

Tryptophan synthase, an α2β2 complex, is a classic example of an enzyme that is thought to "channel" a metabolic intermediate (indole) from the active site of the a subunit to the active site of the β subunit. We now examine the kinetics of substrate channeling by tryptophan synthase directly by chemical quench-flow and stopped-flow methods. The conversion of indole-3-glycerol phosphate (IGP) to tryptophan at the active site proceeds at a rate of 24 s-1, which is limited by the rate of cleavage of IGP to produce indole (α reaction). In a single turnover experiment monitoring the conversion of radiolabeled IGP to tryptophan, only a trace of indole is detectable (≤1% of the IGP), implying that the reaction of indole to form tryptophan must be quite fast (≥1000 s-1). The rate of reaction of indole from solution is much too slow (40 s-1 under identical conditions) to account for the negligible accumulation of indole in a single turnover. Therefore, the indole produced at the α site must be rapidly channeled to the β site, where it reacts with serine to form tryptophan: channeling and the reaction of indole to form tryptophan must each occur at rates ≥1000 s-1. Steady-state turnover is limited by the slow rate of tryptophan release (8 s-1). In the absence of serine, the cleavage of IGP to indole is limited by a change in protein conformation at a rate of 0.16 s-1. When the αβ reaction is initiated by mixing enzyme with IGP and serine simultaneously, there is a lag in the cleavage of IGP and formation of tryptophan. The kinetics of the lag correspond to the rate of formation of the aminoacrylate in the reaction of serine with pyridoxal phosphate at the β site, measured by stopped-flow methods (45 s-1). There is also a change in protein fluorescence, suggestive of a change in protein conformation, occurring at the same rate. Substitution of cysteine for serine leads to a longer lag in the kinetics of IGP cleavage and a correspondingly slower rate of formation of the aminoacrylate (6 s-1). Thus, the reaction of serine at the β site modulates the α reaction such that the formation of the aminoacrylate leads to a change in protein conformation that is transmitted to the α site to enhance the rate of IGP cleavage 150-fold. Analysis of a mutant in the β subunit (E109D) shows that the rate of reaction of indole at the β site is decreased 300-fold, supporting the postulated role of Glu-109 in deprotonating indole to facilitate its reaction with the aminoacrylate. Kinetic analysis of the conversion of IGP and serine to tryptophan catalyzed by the mutant enzyme demonstrates the formation and decay of a significant fraction of indole in a single turnover due to the slower rate of condensation of indole with the aminoacrylate. Taken together, these data demonstrate directly that efficient channeling of indole is a consequence of three features of the reaction kinetics. 1) The rate of diffusion of indole through the channel is very fast; 2) the reaction of indole to form tryptophan at the β site is fast and largely irreversible; and 3) the reaction of serine at the β site modulates the formation of indole at the α site or in the tunnel such that indole is not produced until serine has reacted with pyridoxal phosphate to form the highly reactive aminoacrylate. This intersubunit communication keeps the α and β reactions in phase so that indole does not accumulate at the α site. It is only the combination of these three aspects of the reaction kinetics that leads to efficient channeling of the indole by maintaining a low concentration of indole bound to the enzyme.