title: Mechanism of O-acetylserine sulfhydrylase fromSalmonella typhimurium LT-2.
abstract: O-Acetylserine sulfhydrylase is a pyridoxal 5'-phosphate (PLP) dependent enzyme that catalyzes the final step of L-cysteine biosynthesis inSalmonella, viz. the conversion of O-acetyl-L-serine (OAS) and sulfide to L-cysteine and acetate. A spectrophotometric assay is available using 5-thio(2-nitrobenzoate) (TNB) as an analog of sulfide and monitoring the disappearance of absorbance at 412 nm. The enzyme catalyzes a ping pong mechanism withα-aminoacrylate in Schiff base with the active site PLP as a covalent intermediate. Using data obtained from the pH dependence of kinetic parameters, the acid-base chemical mechanism and the optimum protonation state of enzyme and substrate functional groups necessary for binding has been determined. The Schiff base and theα-amine of the substrate OAS are unprotonated for binding. There also appears to be a requirement for one active site general base to accept a proton from theα-amine and to donate a proton to form cysteine. The enzyme also catalyzes an OAS hydrolase activity, and the pH dependence of this reaction suggests that the active site lysine that participated in the Schiff base linkage is protonated to start the second half reaction, and has a pK of about 8.2. The stereochemistry of(3)H-borohydride reduction of the Schiff base in free enzyme has been determined by degradation of the resulting pyridoxyllysine to pyridoxamine and measuring(3)H-release with apo-aspartate aminotransferase. The sequence around the active site lysine is AsnProSerPheSerValLysCysArg.
authors.affiliation: Departments of Biochemistry and Molecular Biology, Texas College of Osteopathic Medicine, 3500 Camp Bowie Boulevard, 76107-2690, Fort Worth, TX, USA.
authors.forename: S R
authors.initials: SR
authors.lastname: Nalabolu
authors.forename: C H
authors.initials: CH
authors.lastname: Tai
authors.forename: K D
authors.initials: KD
authors.lastname: Schnackerz
authors.forename: P F
authors.initials: PF
authors.lastname: Cook