Application of rapid-scanning, stopped-flow spectroscopy to the characterization of intermediates formed in the reactions of l- and d-tryptophan and β-mercaptoethanol with Escherichia coli tryptophan synthase

Drewe, William Frederick; Koerber, Steven C.; Dunn, Michael F.

doi:10.1016/0300-9084(89)90182-x

Cited by 13 publications

(20 citation statements)

References 25 publications

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“…In the second phase, the peak of the internal aldimine at 412 nm decays and shifts to a 420 nm peak, which was assigned to an external aldimine. 37 There is a increase in the peak at 476 nm, which has been assigned previously to a quinonoid intermediate (Figure 5B,C). 37 The third slow phase that we have observed results in an additional small increase in the 476 nm peak (Figure 5B,C).…”

Section: ■ Resultssupporting

confidence: 73%

“…37 There is a increase in the peak at 476 nm, which has been assigned previously to a quinonoid intermediate (Figure 5B,C). 37 The third slow phase that we have observed results in an additional small increase in the 476 nm peak (Figure 5B,C). The fluorescence emission at >475 nm with 410 nm excitation decreases in three phases in the reaction of Trp synthase with L-Trp (Figure S5), as was seen before.…”

Section: ■ Resultssupporting

confidence: 73%

“…The reaction is biphasic, with 1/τ 1 = 4.5 ± 1 s −1 and 1/τ 2 = 0.15 ± 0.03 s −1 , as was observed previously. 37 In the first phase, there is an increase in absorbance at 325 nm, a decrease at 400 nm, and an increase at 478 nm, similar to the reaction of L-Trp (Figure 7B,C). In the second phase, there is a decrease at 478 nm and an increase at 440 nm, with good isosbestic points at 420 and 472 nm.…”

Section: ■ Resultsmentioning

confidence: 61%

“…Reaction of L-Tryptophan with Trp Synthase. Trp synthase exhibits modest changes in the UV−visible spectrum when mixed with 1 mM L-Trp and 50 mM GP in the rapidscanning stopped-flow spectrophotometer (Figure 5A, 28,37,38 ). However, the reaction of L-Trp is much faster than those of OIA and DOA.…”

Section: ■ Resultsmentioning

confidence: 99%

“…The fast phase fits well to eq 5, with k f = 123 ± 8. 37,38 When Trp synthase crystals are soaked with L-Trp and GP, there is electron density from the ligands observed in the active sites. GP in the α-site is a conformation similar to that in the OIA and DOA complexes (Figure S6A).…”

Section: ■ Resultsmentioning

confidence: 99%

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Structural Basis of the Stereochemistry of Inhibition of Tryptophan Synthase by Tryptophan and Derivatives

Phillips

Harris

2021

Biochemistry

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We have examined the reaction of Salmonella enterica serovar typhimurium tryptophan (Trp) synthase α2β2 complex with l-Trp, d-Trp, oxindolyl-l-alanine (OIA), and dioxindolyl-l-alanine (DOA) in the presence of disodium (dl)-α-glycerol phosphate (GP), using stopped-flow spectrophotometry and X-ray crystallography. All structures contained the d-isomer of GP bound at the α-active site. (3S)-OIA reacts with the pyridoxal-5′-phosphate (PLP) of Trp synthase to form a mixture of external aldimine and quinonoid complexes. The α-carboxylate of OIA rotates about 90° to become planar with the PLP when the quinonoid complex is formed, resulting in a conformational change in the loop of residues 110–115. The COMM domain of the Trp synthase-OIA complex is found as a mixture of two conformations. The (3R)-diastereomer of DOA binds about 5-fold more tightly than (3S)-OIA and also forms a mixture of aldimine and quinonoid complexes. DOA forms an additional H-bond between the 3-OH of DOA and βLys-87. l-Trp does not form a covalent complex with the PLP of Trp synthase. However, d-Trp forms a mixture of two external aldimine complexes which differ in the orientation of the α-carboxylate. In one conformation, the α-carboxylate is in the plane of the PLP, while in the other conformation, the α-carboxylate is perpendicular to the PLP plane. These results confirm that the stereochemistry of the transient indolenine quinonoid intermediate in the mechanism of Trp synthase is (3S) and demonstrate the linkage between aldimine and quinonoid reaction intermediates in the β-active site and allosteric communications with the α-active site.

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Section: ■ Resultssupporting

confidence: 73%

Section: ■ Resultssupporting

confidence: 73%

Section: ■ Resultsmentioning

confidence: 61%

Section: ■ Resultsmentioning

confidence: 99%

Section: ■ Resultsmentioning

confidence: 99%

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