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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 <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