Use of biomolecular interaction analysis to elucidate the regulatory mechanism of the cysteine synthase complex from Arabidopsis thaliana.
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.
Berkowitz
Oliver
O
Institute for Plant Genetics and Crop Plant Research (IPK), Dept. Molecular Cell Biology, Corrensstrasse 3, 06466 Gatersleben, Germany.
Wirtz
Markus
M
Wolf
Alexander
A
Kuhlmann
Jürgen
J
Hell
Rüdiger
R
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
D029681:Arabidopsis Proteins; D012694:Serine; D000123:Acetyltransferases; D051050:Serine O-Acetyltransferase; D003547:Cysteine Synthase; C043943:O-acetylserine