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Zeitschriftenartikel

Use of biomolecular interaction analysis to elucidate the regulatory mechanism of the cysteine synthase complex from <i>Arabidopsis thaliana</i>

MPG-Autoren

Wolf,  Alexander
Max Planck Institute of Molecular Physiology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons98707

Kuhlmann,  Jürgen
Sonstige Wissenschaftliche Organisationseinheiten, Max Planck Institute of Molecular Physiology, Max Planck Society;

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Zitation

Berkowitz, O., Wirtz, M., Wolf, A., Kuhlmann, J., & Hell, R. (2002). Use of biomolecular interaction analysis to elucidate the regulatory mechanism of the cysteine synthase complex from <i>Arabidopsis thaliana</i>. Journal of Biological Chemistry, 277(34): 1, pp. 30629-30634. Retrieved from http://www.jbc.org/cgi/reprint/277/34/30629.pdf.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0014-0E22-D
Zusammenfassung
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 <i>Arabidopsis thaliana</i>. 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 (<i>K</i>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 ?M O-acetylserine and strong positive cooperativity for complex dissociation. The equilibrium of interaction was determined using an enzyme activity-based approach and yielded a <i>K</i>m value of 58 ± 7 ?M 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