Effect of mutation of lysine-120, located at the entry to the active site of O-acetylserine sulfhydrylase-A from Salmonella typhimurium

Chia Hui Tai, Wael M. Rabeh, Rong Guan, Klaus D. Schnackerz, Paul F. Cook

Research output: Contribution to journalArticle

Abstract

O-Acetylserine sulfhydrylase catalyzes the final step of the biosynthesis of l-cysteine, the replacement of the β-acetoxy group of O-acetyl-l-serine (OAS) by a thiol. The enzyme undergoes a conformational change to close the site upon formation of the external Schiff base (ESB) with OAS. Mutation of K120 to Q was predicted to destabilize the closed form of the ESB and decrease the rate. The K120Q mutant enzyme was prepared and characterized by UV-visible absorbance, fluorescence, visible CD, and 31P NMR spectral studies, as well as steady state and pre-steady state kinetic studies. Spectra suggest a shift in the tautomeric equilibrium toward the neutral enolimine and an increase in the rate of interconversion of the open and closed forms of the enzyme. A decrease in the rate of both half reactions likely reflects the stabilization of the ESB as a result of the increased rate of equilibration of the open and closed forms of the enzyme along the reaction pathway. Data suggest a role of K120 in helping to stabilize the closed conformation by participating in a new hydrogen bond to the backbone carbonyl of A231.

Original languageEnglish (US)
Pages (from-to)629-637
Number of pages9
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1784
Issue number4
DOIs
StatePublished - Apr 2008

Keywords

  • O-acetylserine sulfhydrylase
  • P-31 NMR
  • Pyridoxal 5-phosphate
  • Site-directed mutagenesis
  • Spectroscopy

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biophysics
  • Biochemistry
  • Molecular Biology

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