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

Journal Article · Tue Jul 16 04:00:00 EDT 1985 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00336a027· OSTI ID:6259015

Drewe, Jr, W F; Dunn, M F

Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy has been used to investigate the UV-visible absorption changes (300-550 nm) that occur in the spectrum of enzyme-bound pyridoxal 5'-phosphate during the reaction of L-serine with the alpha 2 beta 2 and beta 2 forms of Escherichia coli tryptophan synthase. In agreement with previous kinetic studies the reaction with 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). 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 two transient species, one with lambda max congruent to 420 nm and one with lambda max congruent to 460 nm. These species decay in a biphasic process to a complicated final spectrum with lambda max congruent to 350 nm and with a broad envelope of absorbance extending out to approximately 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. These findings identify the serine Schiff base (the external aldimine) as the 420 nm absorbing, highly fluorescent transient; the species with lambda max congruent to 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 two relaxations and yields a quasi-stable species with lambda max = 420 nm, in good agreement with a previous report.

• Research Organization: Univ. of California, Riverside
• OSTI ID: 6259015
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 15; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ABSORPTION SPECTROSCOPY
ALDEHYDES
AMINO ACIDS
AZINES
BACTERIA
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
ELECTROMAGNETIC RADIATION
ENZYMES
ESCHERICHIA COLI
HETEROCYCLIC COMPOUNDS
HYDROXY ACIDS
KINETICS
LABELLED COMPOUNDS
LIGASES
MICROORGANISMS
NEAR ULTRAVIOLET RADIATION
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC OXYGEN COMPOUNDS
OXYGEN COMPOUNDS
PHOSPHATES
PHOSPHORUS COMPOUNDS
PYRIDINES
PYRIDOXAL
RADIATIONS
REACTION KINETICS
SERINE
SPECTROSCOPY
TRITIUM COMPOUNDS
ULTRAVIOLET RADIATION