Homeostatic regulation upon changes of enzyme activities in the Calvin cycle as an example for general mechanisms of flux control. What can we expect from transgenic plants?

In order to explain the mechanisms of Calvin-cycle regulation, the general properties of metabolic systems under homeostatic flux control are analyzed. It is shown that the main characteristic point for an enzyme in such a system can be the value of a sharp transition from some constant homeostatic flux to a limitation by this enzyme. A special method for the quantitative treatment of the experimental dependence of a metabolic flux such as photosynthesis on enzyme content is developed. It is pointed out that reactions close to a thermodynamic equilibrium under normal conditions can considerably limit the homeostatic fluxes with a decrease of the enzyme content. Calvin-cycle enzymes are classified as non-limiting, near-limiting and limiting. The deduced rules for the regulation of a homeostatic metabolic pathway are used to explain the data obtained for transgenic plants with reduced activities of Calvin-cycle enzymes. The role of compensating mechanisms that maintain the photosynthesis rate constant upon the changes of enzyme contents is analyzed for the Calvin cycle. The developed analysis explains the sharp transitions between limiting and non-limiting conditions that can be seen in transgenic plants with reduced content of some Calvin-cycle enzymes, and the limiting role of such reversible enzymes as aldolase, transketolase and others. The attempt is made to predict the properties of plants with increased enzyme contents in the Calvin cycle.     

Isoenzyme expression changes in response to high temperature determine the metabolic regulation of increased glycolytic flux in yeast

Qualitative phenotypic changes are the integrated result of quantitative changes at multiple regulatory levels. To explain the temperature-induced increase of glycolytic flux in fermenting cultures of Saccharomyces cerevisiae, we quantified the contributions of changes in activity at many regulatory levels. We previously showed that a similar temperature increase in glucose-limited cultivations lead to a qualitative change from respiratory to fermentative metabolism, and this change was mainly regulated at the metabolic level. In contrast, in fermenting cells, a combination of different modes of regulation was observed. Regulation by changes in expression and the effect of temperature on enzyme activities contributed much to the increase in flux. Mass spectrometric quantification of glycolytic enzymes revealed that increased enzyme activity did not correlate with increased protein abundance, suggesting a large contribution of post-translational regulation to activity. Interestingly, the differences in the direct effect of temperature on enzyme kinetics can be explained by changes in the expression of the isoenzymes. Therefore, both the interaction of enzyme with its metabolic environment and the temperature dependence of activity are in turn regulated at the hierarchical level.     

Metabolic control at the cytosol–mitochondria interface in different growth phases of CHO cells

Metabolism at the cytosol–mitochondria interface and its regulation is of major importance particularly for efficient production of biopharmaceuticals in Chinese hamster ovary (CHO) cells but also in many diseases. We used a novel systems-oriented approach combining dynamic metabolic flux analysis and determination of compartmental enzyme activities to obtain systems level information with functional, spatial and temporal resolution. Integrating these multiple levels of information, we were able to investigate the interaction of glycolysis and TCA cycle and its metabolic control. We characterized metabolic phases in CHO batch cultivation and assessed metabolic efficiency extending the concept of metabolic ratios. Comparing in situ enzyme activities including their compartmental localization with in vivo metabolic fluxes, we were able to identify limiting steps in glycolysis and TCA cycle. Our data point to a significant contribution of substrate channeling to glycolytic regulation. We show how glycolytic channeling heavily affects the availability of pyruvate for the mitochondria. Finally, we show that the activities of transaminases and anaplerotic enzymes are tailored to permit a balanced supply of pyruvate and oxaloacetate to the TCA cycle in the respective metabolic states. We demonstrate that knowledge about metabolic control can be gained by correlating in vivo metabolic flux dynamics with time and space resolved in situ enzyme activities.     

Quantifying Dynamic Regulation in Metabolic Pathways with Nonparametric Flux Inference

One of the central tasks in systems biology is to understand how cells regulate their metabolism. Hierarchical regulation analysis is a powerful tool to study this regulation at the metabolic, gene-expression, and signaling levels. It has been widely applied to study steady-state regulation, but analysis of the metabolic dynamics remains challenging because it is difficult to measure time-dependent metabolic flux. Here, we develop a nonparametric method that uses Gaussian processes to accurately infer the dynamics of a metabolic pathway based only on metabolite measurements; from this, we then go on to obtain a dynamical view of the hierarchical regulation processes invoked over time to control the activity in a pathway. Our approach allows us to use hierarchical regulation analysis in a dynamic setting but without the need for explicitly time-dependent flux measurements.     

Control analysis of systems having two steps catalyzed by the same protein molecule in unbranched chains

The analysis of the control of a metabolic pathway having an enzyme catalyzing two different reactions (or a protein displaying two different activities) has been performed. For such systems although the summation theorems are valid, the flux and concentration connectivity theorems of the metabolic control analysis are not valid. Another general relationship of control analysis is shown to be more widely obeyed and holds in these systems. An exemplary case, where the enzyme catalyzes two irreversible reactions, demonstrates that the level of one internal intermediate is constant, i.e. it does not depend upon the independent variables of 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.     

Hierarchical and metabolic regulation of glucose influx in starved Saccharomyces cerevisiae

A novel method dissecting the regulation of a cellular function into direct metabolic regulation and hierarchical (e.g., gene-expression) regulation is applied to yeast starved for nitrogen or carbon. Upon nitrogen starvation glucose influx is down-regulated hierarchically. Upon carbon starvation it is down-regulated both metabolically and hierarchically. The method is expounded in terms of its implications for diverse types of regulation. It is also fine-tuned for cases where isoenzymes catalyze the flux through a single metabolic step.     

Carbon metabolism and the sign of control coefficients in metabolic adaptations underlying K-ras transformation

Metabolic adaptations are associated with changes in enzyme activities. These adaptations are characterized by patterns of positive and negative changes in metabolic fluxes and concentrations of intermediate metabolites. Knowledge of the mechanism and parameters governing enzyme kinetics is rarely available. However, the signs-increases or decreases-of many of these changes can be predicted using the signs of metabolic control coefficients. These signs require the only knowledge of the structure of the metabolic network and a limited qualitative knowledge of the regulatory dependences, which is widely available for carbon metabolism. Here, as a case study, we identified control coefficients with fixed signs in order to predict the pattern of changes in key enzyme activities which can explain the observed changes in fluxes and concentrations underlying the metabolic adaptations in oncogenic K-ras transformation in NIH-3T3 cells. The fixed signs of control coefficients indicate that metabolic changes following the oncogenic transformation-increased glycolysis and oxidative branch of the pentose-phosphate pathway, and decreased concentration in sugar-phosphates-could be associated with increases in activity for glucose-6-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase, and decrease for transketolase. These predictions were validated experimentally by measuring specific activities. We conclude that predictions based on fixed signs of control coefficients are a very robust tool for the identification of changes in enzyme activities that can explain observed metabolic adaptations in carbon metabolism.     

Defining control coefficients in non-ideal metabolic pathways

The extent to which an enzyme controls a flux has been defined as the effect on that flux of a small modulation of the activity of that enzyme divided by the magnitude of the modulation. We here show that in pathways with metabolic channelling or high enzyme concentrations and conserved moieties involving both enzymic and non-enzymic species, this definition is ambiguous; the magnitude of the corresponding flux control coefficient depends on how the enzyme activity is modulated. This is illustrated with two models of biochemically relevant pathways, one in which dynamic metabolite channelling plays a role, and one with a moiety-conserved cycle. To avoid such ambiguity, we view biochemical pathways in a more detailed manner, i.e., as a network of elemental steps. We define 'elemental control coefficients' in terms of the effect on a flux of an equal modulation of the forward and reverse rate constant of any such elemental step (which may correspond to transitions between enzyme states). This elemental control coefficient is independent of the method of modulation. We show how metabolic control analysis can proceed when formulated in terms of the elemental control coefficients and how the traditional control coefficients are related to these elemental control coefficients. An 'impact' control coefficient is defined which quantifies the effect of an activation of all elemental processes in which an enzyme is involved. It equals the sum of the corresponding elemental control coefficients. In ideal metabolic pathways this impact control coefficient reduces to the traditional flux control coefficient. Differences between the traditional control coefficients are indicative of non-ideality of a metabolic pathway, i.e. of channelling or high enzyme concentrations.     

Major metabolic pathways and hormone production in unstimulated monolayer cultures of the rat anterior pituitary

In cell cultures derived from anterior pituitary glands of rats, enzyme activities of cell homogenates and hormone (GH, PRL, LH, and FSH) content of the culture media were measured. Sex differences in enzyme activities representing major metabolic pathways (citrate cycles, pentose cycles, and glycolysis) were demonstrated both in freshly dispersed cells and in 8-day-old cultures; in cultures of both sexes enzyme activities increased during cultivation. In cultures derived from female rats, cell protein doubled by the 12th day and remained constant for up to the 24th day in culture, whereas enzyme activities showed changes suggesting that cell metabolism shifted to anaerobic glycolysis during cultivation. In the culture media the presence of four pituitary hormones was demonstrated for as long as 3 weeks of cultivation with variable secretion dynamics; the release of gonadotropic hormones diminished gradually whereas that of GH remained constant and PRL levels increased with time. These results indicate that under strictly defined culture conditions pituitary cells may function in spite of profound metabolic changes.     

Major metabolic pathways and hormone production in unstimulated monolayer cultures of the rat anterior pituitary

Summary In cell cultures derived from anterior pituitary glands of rats, enzyme activities of cell homogenates and hormone (GH, PRL, LH, and FSH) content of the culture media were measured. Sex differences in enzyme activities representing major metabolic pathways (citrate cycles, pentose cycles, and glycolysis) were demonstrated both in freshly dispersed cells and in 8-day-old cultures; in cultures of both sexes enzyme activities increased during cultivation. In cultures derived from female rats, cell protein doubled by the 12th day and remained constant for up to the 24th day in culture, whereas enzyme activities showed changes suggesting that cell metabolism shifted to anaerobic glycolysis during cultivation. In the culture media the presence of four pituitary hormones was demonstrated for as long as 3 weeks of cultivation with variable secretion dynamics; the release of gonadotropic hormones diminished gradually whereas that of GH remained constant and PRL levels increased with time. These results indicate that under strictly defined culture conditions pituitary cells may function in spite of profound metabolic changes.