title: Use of biomolecular interaction analysis to elucidate the regulatory mechanism of the cysteine synthase complex from Arabidopsis thaliana.
abstract: Real time biomolecular interaction analysis based on surface plasmon resonance has been proven useful for studying protein-protein interaction but has not been extended so far to investigate enzyme-enzyme interactions, especially as pertaining to regulation of metabolic activity. We have applied BIAcore technology to study the regulation of enzyme-enzyme interaction during mitochondrial cysteine biosynthesis in Arabidopsis thaliana. The association of the two enzyme subunits in the hetero-oligomeric cysteine synthase complex was investigated with respect to the reaction intermediate and putative effector O-acetylserine. We have determined an equilibrium dissociation constant of the cysteine synthase complex (K(D) = 25 +/- 4 x 10(-9) m), based on a reliable A + B <--> AB model of interaction. Analysis of dissociation kinetics in the presence of O-acetylserine revealed a half-maximal dissociation rate at 77 +/- 4 microm O-acetylserine and strong positive cooperativity for complex dissociation. The equilibrium of interaction was determined using an enzyme activity-based approach and yielded a K(m) value of 58 +/- 7 microm O-acetylserine. Both effector concentrations are in the range of intracellular O-acetylserine fluctuations and support a functional model that integrates effector-driven cysteine synthase complex dissociation as a regulatory switch for the biosynthetic pathway. The results show that BIAcore technology can be applied to obtain quantitative kinetic data of a hetero-oligomeric protein complex with enzymatic and regulatory function.
authors.affiliation: Institute for Plant Genetics and Crop Plant Research (IPK), Dept. Molecular Cell Biology, Corrensstrasse 3, 06466 Gatersleben, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Berkowitz
authors.forename: Markus
authors.initials: M
authors.lastname: Wirtz
authors.forename: Alexander
authors.initials: A
authors.lastname: Wolf
authors.forename: Jürgen
authors.initials: J
authors.lastname: Kuhlmann
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Hell
mesh_terms: D000123:Acetyltransferases / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry*; D003547:Cysteine Synthase / Q000737:chemistry* / Q000502:physiology; D007700:Kinetics; D012694:Serine / Q000031:analogs & derivatives* / Q000378:metabolism; D051050:Serine O-Acetyltransferase
chemical_list: D029681:Arabidopsis Proteins; D012694:Serine; D000123:Acetyltransferases; D051050:Serine O-Acetyltransferase; D003547:Cysteine Synthase; C043943:O-acetylserine