Mechanism of the addition half of the O-acetylserine sulfhydrylase-A reaction

Wael Rabeh, Susan S. Alguindigue, Paul F. Cook

Research output: Contribution to journalArticle

Abstract

O-Acetylserine sulfhydrylase (OASS) catalyzes the last step in the cysteine biosynthetic pathway in enteric bacteria and plants, substitution of the β-acetoxy group of O-acetyl-L-serine (OAS) with inorganic bisulfide. The first half of the sulfhydrylase reaction, formation of the α-aminoacrylate intermediate, limits the overall reaction rate, while in the second half-reaction, with bisulfide as the substrate, chemistry is thought to be diffusion-limited. In order to characterize the second half-reaction, the pH dependence of the pseudo-first-order rate constant for disappearance of the α-aminoacrylate intermediate was measured over the pH range 6.0-9.5 using the natural substrate bisulfide, and a number of nucleophilic analogues. The rate is pH-dependent for substrates with a pKa > 7, while the rate constant is pH-independent for substrates with a pKa < 7 suggesting that the pKaS of the substrate and an enzyme group are important in this half of the reaction. In D2O, at low pD values, the amino acid external Schiff base is trapped, while in H2O the reaction proceeds through release of the amino acid product, which is likely rate-limiting for all nucleophilic reactants. A number of new β-substituted amino acids were produced and characterized by 1H NMR spectroscopy.

Original languageEnglish (US)
Pages (from-to)5541-5550
Number of pages10
JournalBiochemistry
Volume44
Issue number14
DOIs
StatePublished - Apr 12 2005

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Cysteine Synthase
Substrates
Amino Acids
Rate constants
Schiff Bases
Biosynthetic Pathways
Enterobacteriaceae
Serine
Cysteine
Magnetic Resonance Spectroscopy
Nuclear magnetic resonance spectroscopy
Reaction rates
Bacteria
Substitution reactions
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mechanism of the addition half of the O-acetylserine sulfhydrylase-A reaction. / Rabeh, Wael; Alguindigue, Susan S.; Cook, Paul F.

In: Biochemistry, Vol. 44, No. 14, 12.04.2005, p. 5541-5550.

Research output: Contribution to journalArticle

Rabeh, Wael ; Alguindigue, Susan S. ; Cook, Paul F. / Mechanism of the addition half of the O-acetylserine sulfhydrylase-A reaction. In: Biochemistry. 2005 ; Vol. 44, No. 14. pp. 5541-5550.
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