Serine acetyltransferase of Escherichia coli: substrate specificity and feedback control by cysteine.
Although SAT (serine acetyltransferase) of Escherichia coli, which catalyses the first step in cysteine synthesis, proceeds via a random-order ternary complex reaction mechanism [Hindson and Shaw (2003) Biochemistry 42, 3113-3119], it has been suggested that the nearly identical enzyme from Salmonella typhimurium might involve an acetyl-enzyme intermediate [Leu and Cook (1994) Protein Peptide Lett. 1, 157-162]. In this study the alternative acetyl acceptor threonine and the alternative acyl donor, propionyl-CoA were used to further investigate the reaction mechanism of SAT from E. coli. Steady-state kinetic data and dead-end inhibition studies were again diagnostic of a random-order ternary complex reaction mechanism for alternative substrates. Since earlier kinetic studies with SAT from S. typhimurium suggested that cysteine competes with acetyl-CoA for binding, rather than serine with which it is isostructural, the specificity of the serine-binding pocket was assessed with three substrate mimics; beta-hydroxypropionic acid, glycine and ethanolamine. The data show that SAT interacts productively with the amino and hydroxymethyl moieties of serine, whereas the carboxyl group provides an essential contribution to binding strongly, supporting a view that cysteine will interact productively at the serine-binding site. Furthermore, since the hydroxymethyl contact region of the serine-binding site appears able to accommodate the methylene and acetyl moeties of threonine and O -acetyl-serine respectively, the site is unlikely to provide obligatory short-range contacts with the hydroxyl group of serine, a prerequisite for exclusion of cysteine. Such a proposal is supported by the results of micro-calorimetric studies which show that cysteine competes with serine for binding to SAT rather than with CoA. It follows that tight binding of cysteine at the serine-binding site near the catalytic centre may be the effector of a substantial reduction in the affinity of SAT for CoA, yielding the observed pattern of steady-state inhibition and the mechanism by which cysteine mediates effective end-product control of its synthesis.
Hindson
V John
VJ
Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK. mqbssvjh@fs1.scg.man.ac.uk
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000123:Acetyltransferases / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D002151:Calorimetry / Q000379:methods; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D025461:Feedback, Physiological; D005998:Glycine / Q000378:metabolism; D007700:Kinetics; D012694:Serine / Q000378:metabolism; D051050:Serine O-Acetyltransferase; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism
D000214:Acyl Coenzyme A; D013912:Threonine; C009061:propionyl-coenzyme A; D012694:Serine; D000105:Acetyl Coenzyme A; D000255:Adenosine Triphosphate; D000123:Acetyltransferases; D051050:Serine O-Acetyltransferase; D003545:Cysteine; D005998:Glycine