Characterization of the reaction of L-serine and indole with Escherichia coli tryptophan synthase via rapid-scanning ultraviolet-visible spectroscopy

Abstract

The pre-steady-state reaction of indole and L-serine with the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase has been investigated under different premixing conditions with rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy for the spectral range 300-550 nm. When alpha 2 beta 2 was mixed with indole and L-serine, the reaction of alpha 2 beta 2 was found to occur in three detectable relaxations (1/tau 1 greater than 1/tau 2 greater than 1/tau 3) with rate constants identical with the three relaxations seen in the partial reaction with L-serine [Drewe, W.F., Jr., & Dunn, M.F. (1985) Biochemistry 24, 3977-3987]. Kinetic isotope effects due to substitution of 2H for the alpha-1H of serine were found to be similar to the effects observed in the reaction with serine only. The observed spectral changes and isotope effects indicate that the aldimine of L-serine and PLP and the first quinoid derived from this external aldimine are transient species that accumulate during tau 1. Conversion of these intermediates to the alpha-aminoacrylate Schiff base during tau 2 and tau 3 limits the rate of formation of the second quinoidal species (lambda max 476 nm) generated via C-C bond formation between indole and the alpha-aminoacrylate intermediate. The pre-steady-state reaction of the alpha 2 beta 2-serine mixture with indole is comprised of four relaxations (1/tau 1* greater than 1/tau 2* greater than 1/tau 3* greater than 1/tau 4*).(ABSTRACT TRUNCATED AT 250 WORDS)

pH Dependence of Tryptophan Synthase Catalytic Mechanism

“…2d) at pH values between 6 and 8.8. The kinetics were well fitted to a biexponential process (Table I), as reported previously (22,54). Actually, there is a third very slow phase, characterized by a small amplitude, that we neglected, considering it to be catalytically irrelevant, in agreement with other studies (19,22,30).…”

“…1, j-l), was first monitored by rapid scanning stopped flow to detect simultaneously all forming and decaying catalytic intermediates. Such an informative approach was pioneered by Dunn and co-workers (22,54). The external aldimine with L-serine, absorbing at 422 nm, is formed in 20 -30 ms (Fig.…”

Effect of pH and Monovalent Cations on the Formation of Quinonoid Intermediates of the Tryptophan Synthase α2β2 Complex in Solution and in the Crystal

“…This finding suggests that the equilibrium between E(Aex 1 ) and E(A-A) shifts toward the latter when indoline binds. The binding of indole (8) or indole analogs (11) is known to induce a redistribution of intermediates bound to the ␤-active site.…”

“…Indole is formed from the cleavage of indole-3-glycerol phosphate in the ␣-active site and subsequently is channeled, via a hydrophobic tunnel, to the ␤-active site (5,6) where it is combined with L-Ser to make L-Trp. The reaction at the ␤-active site depends on the cofactor pyridoxal 5Ј-phosphate and proceeds through the formation of several intermediates, which are characterized by distinct absorption properties (Scheme 1) (2,(7)(8)(9)(10).…”

“…The last catalytic intermediate awaiting structural determination is the quinonoid E(Q 3 ) (Scheme 1), formed in the reaction of the E(A-A) with indole. In solution, this species is formed transiently during the ␤-reaction (8,27) or in low amounts upon reaction of the enzyme with the product L-tryptophan (28). In the crystal, this quinonoid does not accumulate appreciably (29).…”