title: A two-step process controls the formation of the bienzyme cysteine synthase complex.
abstract: The regulation of enzyme activity through the transient formation of multiprotein assemblies plays an important role in the control of biosynthetic pathways. One of the first regulatory complexes to be discovered was cysteine synthase (CS), formed by the pyridoxal 5'-phosphate-dependent enzyme O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT). These enzymes are at the branch point of the sulfur, carbon, and nitrogen assimilation pathways. Understanding the mechanism of complex formation helps to clarify the role played by CS in the regulation of sulfur assimilation in bacteria and plants. To this goal, stopped-flow fluorescence spectroscopy was used to characterize the interaction of SAT with OASS, at different temperatures and pH values, and in the presence of the physiological regulators cysteine and bisulfide. Results shed light on the mechanism of complex formation and regulation, so far poorly understood. Cysteine synthase assembly occurs via a two-step mechanism involving rapid formation of an encounter complex between the two enzymes, followed by a slow conformational change. The conformational change likely results from the closure of the active site of OASS upon binding of the SAT C-terminal peptide. Bisulfide, the second substrate and a feedback inhibitor of OASS, stabilizes the CS complex mainly by decreasing the back rate of the isomerization step. Cysteine, the product of the OASS reaction and a SAT inhibitor, slightly affects the kinetics of CS formation leading to destabilization of the complex.
authors.affiliation: Dipartimento di Biochimica e Biologia Molecolare, Università di Parma, 43100 Parma, Italy.
authors.forename: Enea
authors.initials: E
authors.lastname: Salsi
authors.forename: Barbara
authors.initials: B
authors.lastname: Campanini
authors.forename: Stefano
authors.initials: S
authors.lastname: Bettati
authors.forename: Samanta
authors.initials: S
authors.lastname: Raboni
authors.forename: Steven L
authors.initials: SL
authors.lastname: Roderick
authors.forename: Paul F
authors.initials: PF
authors.lastname: Cook
authors.forename: Andrea
authors.initials: A
authors.lastname: Mozzarelli
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D003547:Cysteine Synthase / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D006193:Haemophilus influenzae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D010944:Plants / Q000201:enzymology; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D051050:Serine O-Acetyltransferase / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013455:Sulfur / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D013455:Sulfur; D051050:Serine O-Acetyltransferase; D003547:Cysteine Synthase