Intermediate Trapping via a Conformational Switch in the Na+-Activated Tryptophan Synthase Bienzyme Complex
| Autor: | Michael F. Dunn, Rodney M. Harris |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Indole test
chemistry.chemical_classification biology Protein Conformation Stereochemistry Sodium Substrate (chemistry) Tryptophan synthase Cleavage (embryo) Biochemistry Reversible reaction Amino acid Enzyme Activation Kinetics chemistry.chemical_compound Allosteric Regulation chemistry Catalytic cycle Indoline Tryptophan Synthase biology.protein Nuclear Magnetic Resonance Biomolecular |
| Zdroj: | Biochemistry. 41:9982-9990 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi0255672 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|