The logic of kinetic regulation in the thioredoxin system

Background  

The thioredoxin system consisting of NADP(H), thioredoxin reductase and thioredoxin provides reducing equivalents to a large and diverse array of cellular processes. Despite a great deal of information on the kinetics of individual thioredoxin-dependent reactions, the kinetic regulation of this system as an integrated whole is not known. We address this by using kinetic modeling to identify and describe kinetic behavioral motifs found within the system.     

The regulatory design of an allosteric feedback loop: the effect of saturation by pathway substrate

Aspects of metabolic regulation can be fruitfully studied with a combination of generic modelling, control analysis and graphical analysis using rate characteristics. This paper analyses a prototypical supply-demand system consisting of a biosynthetic subsystem subject to allosteric inhibition by its product and a demand process that consumes this product. The effect of changes in affinity of the committing supply enzyme for the pathway substrate on the regulatory properties of the supply subsystem is compared for the Monod-Wyman-Changeux and the reversible Hill allosteric enzyme models. We found that the Hill model has a distinct advantage in that the steady-state concentration at which it maintains the product is set by the half-saturating product concentration and is independent of changes in the degree of saturation for substrate. In contrast, with the Monod-Wyman-Changeux model this set point varies with affinity for substrate. Explicitly incorporating reversibility in all rate equations made it possible to distinguish between kinetic and thermodynamic aspects of regulation. Combining the supply and demand rate characteristics allows us to explore both the control distribution at steady state and the regulatory performance of the system over a wide range of demand activities.     

Mechanisms of protein-ligand association and its modulation by protein mutations

Protein-ligand interactions are essential for nearly all biological processes, and yet the biophysical mechanism that enables potential binding partners to associate before specific binding occurs remains poorly understood. Fundamental questions include which factors influence the formation of protein-ligand encounter complexes, and whether designated association pathways exist. To address these questions, we developed a computational approach to systematically analyze the complete ensemble of association pathways. Here, we use this approach to study the binding of a phosphate ion to the Escherichia coli phosphate-binding protein. Various mutants of the protein are considered, and their effects on binding free-energy profiles, association rates, and association pathway distributions are quantified. The results reveal the existence of two anion attractors, i.e., regions that initially attract negatively charged particles and allow them to be efficiently screened for phosphate, which is subsequently specifically bound. Point mutations that affect the charge on these attractors modulate their attraction strength and speed up association to a factor of 10 of the diffusion limit, and thus change the association pathways of the phosphate ligand. It is demonstrated that a phosphate that prebinds to such an attractor neutralizes its attraction effect to the environment, making the simultaneous association of a second phosphate ion unlikely. This study suggests ways in which structural properties can be used to tune molecular association kinetics so as to optimize the efficiency of binding, and highlights the importance of kinetic properties.     

Thermostable RNase P RNAs lacking P18 identified in the Aquificales

The RNase P RNA (rnpB) and protein (rnpA) genes were identified in the two Aquificales Sulfurihydrogenibium azorense and Persephonella marina. In contrast, neither of the two genes has been found in the sequenced genome of their close relative, Aquifex aeolicus. As in most bacteria, the rnpA genes of S. azorense and P. marina are preceded by the rpmH gene coding for ribosomal protein L34. This genetic region, including several genes up- and downstream of rpmH, is uniquely conserved among all three Aquificales strains, except that rnpA is missing in A. aeolicus. The RNase P RNAs (P RNAs) of S. azorense and P. marina are active catalysts that can be activated by heterologous bacterial P proteins at low salt. Although the two P RNAs lack helix P18 and thus one of the three major interdomain tertiary contacts, they are more thermostable than Escherichia coli P RNA and require higher temperatures for proper folding. Related to their thermostability, both RNAs include a subset of structural idiosyncrasies in their S domains, which were recently demonstrated to determine the folding properties of the thermostable S domain of Thermus thermophilus P RNA. Unlike 16S rRNA phylogeny that has placed the Aquificales as the deepest lineage of the bacterial phylogenetic tree, RNase P RNA-based phylogeny groups S. azorense and P. marina with the green sulfur, cyanobacterial, and delta/epsilon proteobacterial branches.     

ECA: control in ecosystems

Although metabolic control analysis (MCA) cannot be applied directly to microbial ecological systems because of mass conservation and stoichiometric constraints, we demonstrate here that Hierarchical Control Analysis (HCA) can be applied to such systems. We illustrate the approach for a particular ecosystem example of the biological synthesis of acetic acid from glucose, and uncover some surprising aspects to the control of this miniature ecosystem.     

Sensitivity of Hyperdense Basilar Artery Sign on Non-Enhanced Computed Tomography

Purpose

The hyperdense basilar artery sign (HBAS) is an indicator of vessel occlusion on non contrast-enhanced computer tomography (NECT) in acute stroke patients. Since basilar artery occlusion (BAO) is associated with a high mortality and morbidity, its early detection is of great clinical value. We sought to analyze the influence of density measurement as well as a normalized ratio of Hounsfield unit/hematocrit (HU/Hct) ratio on the detection of BAO on NECT in patients with suspected BAO.

Materials and Methods

102 patients with clinically suspected BAO were examined with NECT followed immediately by Multidetector computed tomography Angiography. Two observers independently analyzed the images regarding the presence or absence of HBAS on NECT and performed HU measurements in the basilar artery. Receiver operating characteristic curve analysis was performed to determine the optimal density threshold for BAO using attenuation measurements or HU/Hct ratio.

Results

Sensitivity of visual detection of the HBAS on NECT was relatively low 81% (95%-CI, 54–95%) while specificity was high 91% (95%-CI, 82–96%). The highest sensitivity was achieved by the combination of visual assessment and additional quantitative attenuation measurements applying a cut-off value of 46.5 HU with 94% sensitivity and 81% specificity for BAO. A HU/Hct ratio >1.32 revealed sensitivity of 88% (95%-CI, 60–98%) and specificity of 84% (95%-CI, 74–90%).

Conclusion

In patients with clinically suspected acute BAO the combination of visual assessment and additional attenuation measurement with a cut-off value of 46.5 HU is a reliable approach with high sensitivity in the detection of BAO on NECT.     

Kinetic model of sucrose accumulation in maturing sugarcane culm tissue

Biochemically, it is not completely understood why or how commercial varieties of sugarcane (Saccharum officinarum) are able to accumulate sucrose in high concentrations. Such concentrations are obtained despite the presence of sucrose synthesis/breakdown cycles (futile cycling) in the culm of the storage parenchyma. Given the complexity of the process, kinetic modelling may help to elucidate the factors governing sucrose accumulation or direct the design of experimental optimisation strategies. This paper describes the extension of an existing model of sucrose accumulation (Rohwer, J.M., Botha, F.C., 2001. Analysis of sucrose accumulation in the sugar cane culm on the basis of in vitro kinetic data. Biochem. J. 358, 437-445) to account for isoforms of sucrose synthase and fructokinase, carbon partitioning towards fibre formation, and the glycolytic enzymes phosphofructokinase (PFK), pyrophosphate-dependent PFK and aldolase. Moreover, by including data on the maximal activity of the enzymes as measured in different internodes, a growth model was constructed that describes the metabolic behaviour as sugarcane parenchymal tissue matures from internodes 3-10. While there was some discrepancy between modelled and experimentally determined steady-state sucrose concentrations in the cytoplasm, steady-state fluxes showed a better fit. The model supports a hypothesis of vacuolar sucrose accumulation against a concentration gradient. A detailed metabolic control analysis of sucrose synthase showed that each isoform has a unique control profile. Fructose uptake by the cell and sucrose uptake by the vacuole had a negative control on the futile cycling of sucrose and a positive control on sucrose accumulation, while the control profile for neutral invertase was reversed. When the activities of these three enzymes were changed from their reference values, the effects on futile cycling and sucrose accumulation were amplified. The model can be run online at the JWS Online database (http://jjj.biochem.sun.ac.za/database/uys).     

Identifying and characterising regulatory metabolites with generalised supply-demand analysis

We present the framework of generalised supply-demand analysis (SDA) of a kinetic model of a cellular system, which can be applied to networks of arbitrary complexity. By fixing the concentrations of each of the variable species in turn and varying them in a parameter scan, rate characteristics of supply-demand are constructed around each of these species. By inspecting the shapes of the rate characteristic patterns and comparing the flux-response coefficients of the supply and demand blocks with the elasticities of the enzymes that interact directly with the fixed metabolite, regulatory metabolites in the system can be identified and characterised. The analysis provides information on whether and where the system is functionally differentiated and which of its species are homeostatically buffered. The novelty in our proposed method lies in the fact that all metabolites are considered for SDA (hence the term “generalised”), which removes investigator bias. It supplies an entry point for the further analysis and detailed characterisation of large models of cellular systems, in which the choice of metabolite around which to perform a SDA is not always obvious.