Detection and identification of intermediates in the reaction of L-serine with Escherichia coli tryptophan synthase via rapid-scanning ultraviolet-visible spectroscopy

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Abstract
Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy was used to investigate the UV-visible absorption changes (300-550 nm) that occur in the spectrum of enzyme-bound pyridoxal 5''-phosphate (PLP) during the reaction of L-serine withthe .alpha.2.beta.2 and .beta.2 forms of E. coli tryptophan synthase. In agreement with previous kinetic studies the reaction with .alpha.2.beta.2 was found to occur in 3 detectable relaxations (1/.tau.1 > 1/.tau.2 > 1/.tau.3). The RSSF data reveal that during .tau.1, the internal aldimine, E(PLP), with .lambda.max = 412 nm (pH 7.8), undergoes rapid conversion to 2 transient species, 1 with .lambda.max .simeq. 420 nm and 1 with .lambda.max .apprx. 460 nm. These species decay in a biphasic process (1/.tau.2, 1/.tau.3) to a complicated final spectrum with .lambda.max .simeq. 350 nm and with a broad envelope of absorbance extending out to .apprx. 525 nm. Analysis of the time-resolved spectra establishes that the spectral changes in .tau.2 are nearly identical with the spectral changes in .tau.3. Kinetic isotope effects due to substitution of 2H for the .alpha.-1H of serine were found to increase the amount of the 420-nm transient and to decrease the amount of the species with .lambda.max .simeq. 460 nm. These findings identify the serine Schiff base (the external aldimine) as the 420 nm absorbing, highly fluorescent transient; the species with .lambda.max .simeq. 460 nm is the delocalized carbanion (quinoidal) species derived from abstraction of the .alpha. proton from the external aldimine. The reaction of L-serine with .beta.2 consists of 2 relaxations (1/.tau.1.beta. > 1/.tau.2.beta.) and yields a quasi-stable species with .lambda.max = 420 nm, in good agreement with a previous report. Analysis of the RSSF spectra indicates that the same spectral change occurs in each phase of the reaction. The similarity of the spectral changes that occur in .tau.2 and .tau.3 of the .alpha.2.beta.2 reaction is postulated to originate from the existence of 2 (slowly) interconverting forms of the enzyme. A similar explanation is proposed to explain the biphasic character of the reaction of .beta.2 with L-serine. It is proposed that the spectrum of the product of the L-serine reaction with .alpha.2.beta.2 contains contributions from the Schiff base of the reactive .alpha.-aminoacrylase species, the external aldimine and the quinoidal species, all in rapid equilibrium.