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.

The Structure and Mechanism of Serine Acetyltransferase from Escherichia coli

“…Removal of this interaction could significantly reduce cysteine binding energy. We would argue, based on the structure presented here, the modeling of the tetrahedral intermediate, and on previous biochemical assays (15,16), that it is the serine binding pocket and not the acetyl-CoA binding channel that is occupied by cysteine to inhibit the O-acetylation of serine reaction performed by SAT. The tight binding of cysteine, particularly the interactions of the two histidine side chains from adjacent monomers, may involve a slight distortion of the acetyl-CoA binding site, such that the affinity for the coenzyme is reduced.…”

The Polysialic Acid-specific O-Acetyltransferase OatC from Neisseria meningitidis Serogroup C Evolved Apart from Other Bacterial Sialate O-Acetyltransferases

“…Compared with ␣/␤-hydrolase fold enzymes, members of the hexapeptide repeat family not only adopt an entirely different structural fold, the left-handed ␤-helix fold, but also act by a different kinetic mechanism. Although they possess a strictly conserved catalytic histidine, this residue is not part of a catalytic triad, and kinetic data obtained for this enzyme family revealed a sequential kinetic mechanism (24,62,80,81).…”

Three-stage Assembly of the Cysteine Synthase Complex from Escherichia coli

“…Hydrogen sulfide then adds to the enzyme and attacks the ␤-carbon of the intermediate, forming a cysteine-PLP adduct that is cleaved to release cysteine during re-attachment of PLP to the conserved lysine. The SAT mechanism had long been considered to be ping pong (32,33); however, recent revaluations suggest that it is random sequential (34,35).…”