Summary
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 of3H-borohydride reduction of the Schiff base in free enzyme has been determined by degradation of the resulting pyridoxyllysine to pyridoxamine and measuring3H-release with apo-aspartate aminotransferase. The sequence around the active site lysine is AsnProSerPheSerValLysCysArg.
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References
Becker MA, Kredich NM, Tomkins GM (1969) J Bio Chem 244: 2418–2427
Byrne CR, Monroe RS, Ward KA, Kredich NM (1988) J Bacteriol 170: 3150–3157
Cleland, WW (1977) Adv Enzymol Relat Areas Mol Biol 45: 273–387
Cleland WW (1979) Methods Enzymol 63: 103–138
Cook PF, Wedding RT (1976) J Biol Chem 251: 2023–2029
Cook PF, Wedding RT (1977) J Biol Chem 252: 3459
Dunathan HC (1971) Adv Enzymol 35: 79–134
Dunathan HC, Voet JG (1974) Proc Natl Acad Sci USA 71: 3888–3891
Ellman GL (1959) Arch Biochem Biophys 82: 70–77
Hara S, Payne MA, Schnackerz KD, Cook PF (1990) Protein Expression Purification 1: 70–76
Kredich NM, Tomkins GM (1966) J Biol Chem 241: 4955–4965
Levy S, Danchin A (1988) Mol Microbiol 2: 777–783
Miles EW, Houck DR, Floss HG (1982) J Bio Chem 257: 14203–14210
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Nalabolu, S.R., Tai, C.H., Schnackerz, K.D. et al. Mechanism of O-acetylserine sulfhydrylase fromSalmonella typhimurium LT-2. Amino Acids 2, 119–125 (1992). https://doi.org/10.1007/BF00806082
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DOI: https://doi.org/10.1007/BF00806082