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.
Highlights
The interaction of proteins is essential for the function of living cells [1, 2]
Cysteine Synthase Complex Expression, Purification, and Characterization—Detailed biochemical and biophysical analysis of the interaction of the Serine acetyltransferase (SAT) and OAS-TL subunits of the cysteine synthase complex (CSC) was hampered in the past by limiting protein preparation procedures
To prepare sufficient amounts of purified complex, the co-expression and purification protocol for the mitochondrial CSC consisting of SAT-A and OAS-TL-C was optimized for the recombinant proteins from E. coli and could be completed within 2 h after harvest of cells
Summary
The interaction of proteins is essential for the function of living cells [1, 2]. Monitoring of biomolecular interaction analysis can be achieved by BIAcore technology that is based on surface plasmon resonance (SPR). The physical phenomenon of SPR is widely used to visualize macromolecular interactions in real time with the advantages of no labeling requirements and the option to determine kinetic rate constants. It is mostly applied to quantification of antigen/antibody binding, receptor ligand screening, or characterization of protein modification It has not been used so far to investigate enzyme-enzyme interactions, in particular with respect to regulation of the metabolic activity of multienzyme complexes. In this study we have applied SPR to analyze the interaction of a hetero-oligomeric metabolic protein complex using the enzymes of cysteine biosynthesis from mitochondria of Arabidopsis thaliana. One tetramer of SAT and two dimers of OAS-TL are presumed to form the hetero-oligomeric cysteine synthase complex (CSC) that was first described for Salmonella typhimurium [3, 4] Proteinprotein interactions of such metabolic enzymes mostly serve to channel substrate intermediates between active sites of sequential reaction steps of a pathway. Incubation of the intact CSC with OAS dissociates the complex in vitro and results in changes of kinetic properties and activity of both enzymes; free SAT tends to aggregate, This paper is available on line at http://www.jbc.org
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