Robustness in Glyoxylate Bypass Regulation

Citation: Shinar G, Rabinowitz JD, Alon U ( Robustness in Glyoxylate Bypass Regulation Guy Shinar 0 Joshua D. Rabinowitz 0 Uri Alon 0 Jason A. Papin, University of Virginia, United States of America 0 1 Departments of Molecular Cell Biology and Physics of Complex Systems, Weizmann Institute of Science , Rehovot , Israel , 2 Department of Chemistry and Lewis-Sigler Institute for Integrative Genomics, Princeton University , Princeton, New Jersey , United States of America The glyoxylate bypass allows Escherichia coli to grow on carbon sources with only two carbons by bypassing the loss of carbons as CO2 in the tricarboxylic acid cycle. The flux toward this bypass is regulated by the phosphorylation of the enzyme isocitrate dehydrogenase (IDH) by a bifunctional kinase-phosphatase called IDHKP. In this system, IDH activity has been found to be remarkably robust with respect to wide variations in the total IDH protein concentration. Here, we examine possible mechanisms to explain this robustness. Explanations in which IDHKP works simultaneously as a first-order kinase and as a zero-order phosphatase with a single IDH binding site are found to be inconsistent with robustness. Instead, we suggest a robust mechanism where both substrates bind the bifunctional enzyme to form a ternary complex. - Funding: GS and UA were supported by the Kahn Family Foundation. JDR was supported by the National Science Foundation (Grant MCB-0643859) and the National Institutes of Health (Grant 5 P50 GM071508). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Robustness in biological systems has seen a renewal of research interest in recent years [112]. To define robustness, one needs to specify what feature is robust and with respect to which variations. Classic experimental studies have shown that metabolic fluxes are often insensitive to the levels of enzymes in the pathway, as reviewed in [13]. Metabolic control theory addresses this by suggesting that control of flux is distributed amongst many enzymes, and thus no single enzyme is rate limiting. In the last decade, studies have added a new level of understanding on robustness by providing detailed molecular mechanisms that can preserve the essential function of a system in the face of large variations in the protein levels. For example, specific mechanisms explain how exact adaptation in bacterial chemotaxis is robust with respect to chemotaxis protein levels [2,3], and how patterning in drosophila embryos is robust with respect to morphogen production rates [12,14,15]. A recent review summarizes experiments and theoretical mechanisms for robustness [10]. Recently, an intriguing class of robust mechanisms has been found, based on bifunctional enzymes that carry out two opposing reactions (such as both modifying a target protein, and removing the modification) [8,11]. These robust mechanisms seem to apply to a class of bacterial two-component signaling system. These systems show robustness of input-output relations, in the sense that output responds to input signals in a way that is not disrupted by variations in protein levels. Here, we extend this line of research to one of the best studied regulation steps in E. coli metabolism, the IDHKP/IDH system. This system raised our interest because it employs a bifunctional enzyme that carries out two opposing reactions, hinting at a robust mechanism. However, it has several biochemical differences from previously studied systems [8,11], suggesting that it may show a new type of robust mechanism. The need for precise regulation in the IDHKP/IDH system is evident from its biological function. The IDH system regulates the partitioning of carbon flux between the TCA cycle and the glyoxylate bypass (Figure 1). Precise regulation of flux to the glyoxylate bypass is essential when the bacterium grows on substances such as acetate that contain only two carbon atoms. Without the glyoxylate bypass, both carbon atoms would be converted to CO2 by the TCA cycle, thereby leaving no carbon available for biosynthesis of cell constituents. Hence, growth on acetate and other two-carbon compounds requires directing some of the carbon flux to the glyoxylate bypass, thereby avoiding carbon loss. The precise partitioning of carbon flux between the cycle and the bypass is achieved by regulating the activity of the enzyme IDH (isocitrate dehydrogenase), which stands at the entry to the bypass. The activity of IDH is determined by its phosphorylation state: only unphosphorylated IDH is active. During growth on substances with more than two carbon atoms, IDH is mostly unphosphorylated and hence active. Thus, most of the carbon flux is directed to the more efficient TCA cycle. On the contrary, during growth on acetate, most of IDH is phosphorylated and hence inactive, so that a large part of the carbon flux is directed to the bypass [1619]. To regulate the IDH phosphorylation level, E. coli employs a bifunctional enzyme. This enzyme catalyzes both the phosphorylation of IDH, and its dephosphorylation, and is called IDHKP (IDH Kinase/Phosphatase) [20]. IDHKP uses ATP as the phosphoryl donor for the kinase reaction, and also requires ATP as a cofactor for the dephosphorylation reaction [2022]. The activity of IDHKP is allosterically regulated by the levels of various metabolites in the cell that act as the input signals to this system [21]. The robustness of IDH activity has been experimentally tested by Laporte et. al. [23]. It was found that during growth on acetate, the concentration of active (unphosphorylated) IDH is extremely To grow well, the cell needs to produce a balanced set of building blocks by means of its metabolic network. Regulatory circuits are used to maintain appropriate fluxes as metabolites flow through the branching pathways in the network. Here, we asked how such regulatory circuits can work precisely, despite the fact that they are made of proteins whose levels vary from cell to cell and in the same cell over time. We used a well-studied circuit, at a key branch point called the glyoxylate bypass, as a model system. Previous experiments showed that this system is remarkably robust to changes in the levels of its proteins. Here, we propose a mechanism to explain this robustness, based on a bifunctional enzyme that catalyzes two opposing reactions. We show that a simple explanation based on enzyme saturation is inconsistent with more rigorous mathematical analysis. Our proposed mechanism suggests several experimentally testable predictions. It shows how a systems-level feature (robustness) may arise from seemingly unrelated biochemical details. Because analogous designs with bifunctional enzymes are found in other systems in different organisms, the present mechanism might apply more broadly. robust: The level of active IDH chan (...truncated)