Chemical Probes of Substrate Channeling in The E. Coli Tryptophan Synthase α2β2 Bienzyme Complex

Dunn, Michael F.; Roy, M.; Robustell, Brian; Aguilar, Valentin

doi:10.1007/978-3-0348-9308-4_31

Cited by 11 publications

(8 citation statements)

References 15 publications

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“…The experiments of Anderson et al [66] also provided further evidence showing a functional role for the tunnel in the channeling of indole and further confirming that reaction of L-Ser activates the α-site. We established that the tunnel indeed functions to transfer Indole between the active sites [41–44, 59, 60, 62, 63, 67, 68], and that it is the conversion of the L-Ser external aldimine to the α-aminoacrylate (via the Ser quinonoid intermediate) that activates the α-subunit [61], whereas conversion of the L-Trp quinonoid to the L-Trp external aldimine switches the α-subunit off [63]. These findings led to the proposal that the switching of subunits between open and closed conformations switches the activity of the α-site on and off.…”

Section: Origins Of Allosteric Interactions In Tryptophan Synthase α-mentioning

confidence: 99%

Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex

Dunn

2012

Archives of Biochemistry and Biophysics

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The tryptophan synthase α2β2 bi-enzyme complex catalyzes the last two steps in the synthesis of L-tryptophan (L-Trp). The α-subunit catalyzes cleavage of 3-indole-D-glycerol 3’-phosphate (IGP) to give indole and D-glyceraldehyde 3’-phosphate (G3P). Indole is then transferred (channeled) via an interconnecting 25 Å-long tunnel, from the α-subunit to the (β-subunit where it reacts with L-Ser in a pyridoxal 5’-phosphate-dependent reaction to give L-Trp and a water molecule. The efficient utilization of IGP and L-Ser by tryptophan synthase to synthesize L-Trp utilizes a system of allosteric interactions that (1) function to switch the α-site on and off at different stages of the β-subunit catalytic cycle, and (2) prevent the escape of the channeled intermediate, indole, from the confines of the α- and β-catalytic sites and the interconnecting tunnel. This review discusses in detail the chemical origins of the allosteric interactions responsible both for switching the α-site on and off, and for triggering the conformational changes between open and closed states which prevent the escape of indole from the bienzyme complex.

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Section: Origins Of Allosteric Interactions In Tryptophan Synthase α-mentioning

confidence: 99%

Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex

Dunn

2012

Archives of Biochemistry and Biophysics

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134

224

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“…The combined α- and β-reactions are designated as the αβ-reaction (eq 3). Rapid kinetic studies have shown that indole is channeled through the connecting tunnel between the α- and β-sites ( − ) …”

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Intermediate Trapping via a Conformational Switch in the Na⁺-Activated Tryptophan Synthase Bienzyme Complex

Harris

Dunn

2002

Biochemistry

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The tryptophan synthase bienzyme complex channels substrate indole between the alpha- and beta-sites via a 25 A long interconnecting tunnel. Channeling efficiency is dependent upon a conformational switch in alphabeta-dimeric units between open conformations of low activity to which substrates bind and closed conformations of high activity wherein substrates react. In experiments designed to gain a better understanding of the linkage between chemical steps and conformational transitions in the catalytic cycle, the novel amino acid dihydroiso-L-tryptophan (DIT) was used as an analogue of L-Trp. In the forward reaction (indoline + L-Ser) to synthesize DIT, the quinonoid species, E(Q)(indoline), is formed quickly, while in the reverse reaction (DIT cleavage), the accumulation of E(Q)(indoline) occurs very slowly. Nevertheless, when the alpha-site substrate analogue alpha-D,L-glycerol phosphate (GP) is bound, DIT cleavage was found to give a rapid formation and dissipation of E(Q)(indoline) followed by a very slow reappearance of E(Q)(indoline). This result led to the conclusion that the reaction of DIT proceeds quickly through the quinonoid state to give indoline and the alpha-aminoacrylate Schiff base, E(A-A), both in the absence and in the presence of GP. In the absence of GP the slow conversion of E(A-A) to pyruvate and ammonium ion limits the rate of accumulation of free indoline and therefore the rate of buildup of E(Q)(indoline). However, when GP is bound to the alpha-site, the indoline generated by DIT cleavage in the first turnover is trapped within the enzyme complex, shifting the equilibrium distribution strongly in favor of E(Q)(indoline) as a consequence of the high local concentration of sequestered indoline. This sequestering is the result of a switching of alphabeta-subunit pairs to a closed conformation when GP binds to the alpha-site and E(A-A) and/or E(Q)(indoline) is formed at the beta-site, thereby trapping indoline inside. The decay of the transiently formed E(Q)(indoline) occurs due to leakage of indoline from the closed system.

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“…No allosteric interactions are known to be transferred across the β−β subunit interface of the holoenzyme. The active sites of neighboring α- and β-subunits are separated by about 25−30 Å and are connected by a tunnel ( , ), the preferential route of transfer for the common intermediate, indole, from the α-site to the β-site ( − ). A system of reciprocal allosteric interactions exerted through the α−β subunit interface ensures the coordination of the α- and β-reactions ( 2 , 6 , 16 , 17 , 19 , − (Figure ).…”

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Mechanisms of Monovalent Cation Action in Enzyme Catalysis: The First Stage of the Tryptophan Synthase β-Reaction

1999

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The tryptophan synthase bienzyme complex is activated and regulated by the allosteric action of monovalent cations (MVCs). The kinetic dissection of the first stage (stage I) in the beta-reaction of tryptophan synthase, the reaction of L-serine with pyridoxal 5'-phosphate at the beta-site to give the alpha-aminoacrylate Schiff base intermediate, E(A-A), is here examined in the absence and presence of MVCs. This analysis reveals which of the individual steps are greatly affected in stage I and how the ground states and transition states are affected by MVCs. Kinetic studies in combination with a detailed relaxation kinetic analysis to determine the specific rate constants for the conversion of the L-Ser external aldimine, E(Aex1), to E(A-A) show that the primary kinetic isotope effect for proton abstraction from Calpha of the E(Aex1) species changes from 4.0 +/- 0.4 in the absence of MVCs to a value of 5.9 +/- 0.5 in the presence of Na+, indicating that the nature of the transition state for this C-H scission is significantly perturbed by the MVC effect. The E(A-A) species was found to exist in two conformations with different activities, the ratio of which is affected by the presence of MVCs. It is shown that changes in the rate constants of stage I are important in establishing the ratio of active to inactive conformations of the E(A-A) species. Consequently, the MVC effect alters the relative energies of both the transition states and the ground states for selected steps in stage I of the pathway. Hence, interactions at the MVC site give rise to a fine-tuning of the covalent bonding interactions between active site residues and the reacting substrate during the conformational cycle of the bienzyme complex.

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Chemical Probes of Substrate Channeling in The E. Coli Tryptophan Synthase α2β2 Bienzyme Complex

Cited by 11 publications

References 15 publications

Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex

Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex

Intermediate Trapping via a Conformational Switch in the Na⁺-Activated Tryptophan Synthase Bienzyme Complex

Mechanisms of Monovalent Cation Action in Enzyme Catalysis: The First Stage of the Tryptophan Synthase β-Reaction

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Chemical Probes of Substrate Channeling in The E. Coli Tryptophan Synthase α2β2 Bienzyme Complex

Cited by 11 publications

References 15 publications

Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex

Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex

Intermediate Trapping via a Conformational Switch in the Na+-Activated Tryptophan Synthase Bienzyme Complex

Mechanisms of Monovalent Cation Action in Enzyme Catalysis: The First Stage of the Tryptophan Synthase β-Reaction

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Intermediate Trapping via a Conformational Switch in the Na⁺-Activated Tryptophan Synthase Bienzyme Complex