title: Random-order ternary complex reaction mechanism of serine acetyltransferase from Escherichia coli.
abstract: Although serine acetyltransferase (SAT) from Escherichia coli is homologous with a number of bacterial enzymes that catalyze O-acetyl transfer by a sequential (ternary complex) mechanism, it has been suggested, from experiments with the nearly identical enzyme from Salmonella typhimurium, that the reaction could proceed via an acetyl-enzyme intermediate. To resolve the matter, the E. coli gene for SAT was overexpressed and the enzyme purified 13-fold to homogeneity. The results of a steady-state kinetic analysis of the forward reaction are diagnostic for a ternary complex mechanism, and the response of SAT to dead-end inhibitors indicates a random order for the addition of substrates. The linearity of primary double-reciprocal plots, in the presence and absence of dead-end inhibitors, argues that interconversion of ternary complexes is not significantly faster than kcat, whereas substrate inhibition by serine suggests that breakdown of the SAT.CoA binary complex is rate-determining. The results of equilibrium isotope exchange experiments, for both half-reactions, rule out a "ping-pong" mechanism involving an acetyl-enzyme intermediate, and a pre-steady-state kinetic analysis of the turnover of AcCoA supports such a conclusion. Kinetic data for the reverse reaction (acetylation of CoA by O-acetylserine) are also consistent with a steady-state random-order mechanism, wherein both the breakdown of the SAT*serine complex and the interconversion of ternary complexes are partially rate-determining.
authors.affiliation: Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom. mqbssvjh@fs1.scg.man.ac.uk
authors.forename: V John
authors.initials: VJ
authors.lastname: Hindson
authors.forename: William V
authors.initials: WV
authors.lastname: Shaw
mesh_terms: D000105:Acetyl Coenzyme A / Q000737:chemistry; D000123:Acetyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D001667:Binding, Competitive; D002384:Catalysis; D003065:Coenzyme A / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D004952:Esters; D005998:Glycine / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D010759:Phosphorus Isotopes; D017433:Protein Structure, Secondary; D012694:Serine / Q000737:chemistry; D051050:Serine O-Acetyltransferase; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D004952:Esters; D010759:Phosphorus Isotopes; D012694:Serine; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; D051050:Serine O-Acetyltransferase; D003065:Coenzyme A; D005998:Glycine