title: Acid-base chemical mechanism of O-acetylserine sulfhydrylases-A and -B from pH studies.
abstract: The pH dependence of kinetic parameters using natural and alternative reactants was determined in order to obtain information on the chemical mechanisms of the A and B isozymes of O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium. A general mechanism is proposed for OASS in which OAS binds with its alpha-amine unprotonated to carry out a nucleophilic attack on C4' of the protonated Schiff base and with the acetyl carbonyl hydrogen-bonded to a protonated enzyme group (or a water molecule), which aids in the beta-elimination of acetate. The enzyme lysine that was in Schiff base linkage with the active site pyridoxal 5'-phosphate deprotonates the alpha-carbon in the beta-elimination reaction, and a proton is likely released with the acetate product. Sulfide likely binds as HS- to undergo nucleophilic attack on the alpha-aminoacrylate intermediate, followed by protonation of the alpha-carbon by the enzyme lysine. In OASS-A, HS- is hydrogen-bonded to the enzyme group that assists in the beta-elimination of acetate, but this is not the case for OASS-B. The pH independent equilibrium constant for the first half-reaction of OASS-A is 1.6 x 10(-3), while the second half-reaction is practically irreversible.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of North Texas Health Sciences Center, Fort Worth 76107, USA.
authors.forename: C H
authors.initials: CH
authors.lastname: Tai
authors.forename: S R
authors.initials: SR
authors.lastname: Nalabolu
authors.forename: J W
authors.initials: JW
authors.lastname: Simmons
authors.forename: T M
authors.initials: TM
authors.lastname: Jacobson
authors.forename: P F
authors.initials: PF
authors.lastname: Cook
mesh_terms: D000085:Acetates / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D003547:Cysteine Synthase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008956:Models, Chemical; D009579:Nitrobenzoates; D011732:Pyridoxal Phosphate / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology; D012545:Schiff Bases; D012694:Serine / Q000031:analogs & derivatives / Q000378:metabolism; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds; D013816:Thermodynamics; D017186:Titrimetry
chemical_list: D000085:Acetates; D007527:Isoenzymes; D009579:Nitrobenzoates; D012545:Schiff Bases; D013438:Sulfhydryl Compounds; C011136:thionitrobenzoic acid; D012694:Serine; D011732:Pyridoxal Phosphate; D003547:Cysteine Synthase; C043943:O-acetylserine; D008239:Lysine