Optimization-based metabolic control analysis

In this work, a novel optimization-based metabolic control analysis (OMCA) method is introduced for reducing data requirement for metabolic control analysis (MCA). It is postulated that using the optimal control approach, the fluxes in a metabolic network are correlated to metabolite concentrations and enzyme activities as a state-feedback control system that is optimal with respect to a homeostasis objective. It is then shown that the optimal feedback gains are directly related to the elasticity coefficients (ECs) of MCA. This approach requires determination of the relative "importance" of metabolites and fluxes for the system, which is possible with significantly reduced experimental data, as compared with typical MCA requirements. The OMCA approach is applied to a top-down control model of glycolysis in hepatocytes. It is statistically demonstrated that the OMCA model is capable of predicting the ECs observed experimentally with few exceptions. Further, an OMCA-based model reconciliation study shows that the modification of four assumed stoichiometric coefficients in the model can explain most of the discrepancies, with the exception of elasticities with respect to the NADH/NAD ratio.     

Subtleties in control by metabolic channelling and enzyme organization

Because of its importance to cell function, the free-energy metabolism of the living cell is subtly and homeostatically controlled. Metabolic control analysis enables a quantitative determination of what controls the relevant fluxes. However, the original metabolic control analysis was developed for idealized metabolic systems, which were assumed to lack enzyme-enzyme association and direct metabolite transfer between enzymes (channelling). We here review the recently developed molecular control analysis, which makes it possible to study non-ideal (channelled, organized) systems quantitatively in terms of what controls the fluxes, concentrations, and transit times. We show that in real, non-ideal pathways, the central control laws, such as the summation theorem for flux control, are richer than in ideal systems: the sum of the control of the enzymes participating in a non-ideal pathway may well exceed one (the number expected in the ideal pathways), but may also drop to values below one. Precise expressions indicate how total control is determined by non-ideal phenomena such as ternary complex formation (two enzymes, one metabolite), and enzyme sequestration. The bacterial phosphotransferase system (PTS), which catalyses the uptake and concomitant phosphorylation of glucose (and also regulates catabolite repression) is analyzed as an experimental example of a non-ideal pathway. Here, the phosphoryl group is channelled between enzymes, which could increase the sum of the enzyme control coefficients to two, whereas the formation of ternary complexes could decrease the sum of the enzyme control coefficients to below one. Experimental studies have recently confirmed this identification, as well as theoretically predicted values for the total control. Macromolecular crowding was shown to be a major candidate for the factor that modulates the non-ideal behaviour of the PTS pathway and the sum of the enzyme control coefficients.     

Kinetic properties required for sustained or paradoxical control of metabolic fluxes under large changes in enzyme activities

The effect that an increase in the activity of an enzyme has on its flux normally decreases with activity increase. To achieve a large increase in flux by manipulating a single step would therefore require a high initial effect that maintains or increases when the activity is increased, what has been called sustained or paradoxical control. Using metabolic control analysis for large responses, we derive conditions for sustained or paradoxical control in terms of elasticity coefficients. These are used to characterise types of rate laws contributing to this behaviour. The result that simple pathways, with normal kinetics, subject to large activity changes can lead to paradoxical control behaviour suggests that this type of pattern may be much more ubiquitous than could have, in principle, been suspected.     

Correlation between enzyme activities and routine metabolic rate in Drosophila

To determine whether enzyme activity is correlated with physiological performance, we analysed the relationship between routine metabolic rate and published data on activity of 12 enzymes from nine species of Drosophila. The enzymes are involved in several aspects of intermediary metabolism including glycolysis. Multiple regression on phylogenetically independent contrasts revealed significant and positive correlations between in vitro enzyme activity and routine metabolic rate. The regression analysis included body size and locomotor activity level as covariates. This result suggests that there may be energetic costs associated with increased enzyme capacity.     

Application of the transition time of metabolic system as a criterion for optimization of metabolic processes

Transition time of metabolic systems in introduced as a suitable optimization criterion for biotechnological processes in which it is desirable to reduce the lag time and minimize the mass contained within the system. Lag time is the time needed for the system to attain the steady state. Results obtained from the sensitivity analysis of this steady state response are presented within the metabolic control analysis and applied to 3 case studies. In all of them the information provided by the transition time control profile allows the implementation of a strategy for biotechnological manipulations aimed at the improvement of the process. (c) 1994 John Wiley & Sons, Inc.     

代谢网络定量分析研究进展

综述了代谢工程中代谢控制分析、代谢通量分析、生化系统理论、途径分析、控制论模型等定量分析方法的基本理论,以实例说明了这些方法的应用,并对代谢分析方法的发展进行了展望。     

Modeling with a view to target identification in metabolic engineering: A critical evaluation of the available tools

The state of the art tools for modeling metabolism, typically used in the domain of metabolic engineering, were reviewed. The tools considered are stoichiometric network analysis (elementary modes and extreme pathways), stoichiometric modeling (metabolic flux analysis, flux balance analysis, and carbon modeling), mechanistic and approximative modeling, cybernetic modeling, and multivariate statistics. In the context of metabolic engineering, one should be aware that the usefulness of these tools to optimize microbial metabolism for overproducing a target compound depends predominantly on the characteristic properties of that compound. Because of their shortcomings not all tools are suitable for every kind of optimization; issues like the dependence of the target compound's synthesis on severe (redox) constraints, the characteristics of its formation pathway, and the achievable/desired flux towards the target compound should play a role when choosing the optimization strategy. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

UFLC-MS/MS研究胡椒碱在不同种属肝微粒体中的代谢稳定性和代谢表型

应用体外肝微粒体孵育体系,考察胡椒碱在人、SD大鼠、小鼠、恒河猴和比格犬5个种属肝微粒体中的代谢稳定性,比较代谢的种属差异,确定其在人肝微粒体中的代谢表型。通过UFLC-MS/MS检测方法,测定胡椒碱在各个种属肝微粒体中孵育后的剩余浓度,考察他们的代谢稳定性及体外代谢动力学参数。采用化学抑制法考察胡椒碱在人肝微粒体中的代谢表型。结果表明胡椒碱在人、SD大鼠、小鼠、恒河猴和比格犬的肝微粒体中,半衰期T1/2分别为31. 36、48. 46、138. 60、147. 45、165. 00 min;体外固有清除率CLint分别为0. 0442、0. 0286、0. 0100、0. 0094、0. 0084m L/(m L·mg);在人肝微粒体中,胡椒碱主要被CYP3A4和CYP2C9酶代谢。推测胡椒碱在各种肝微粒体中的代谢均相对较稳定,其中大鼠和人的肝微粒体代谢性质最相近,在后续的实验中可以考虑用大鼠的代谢结果预测人的代谢结果;人肝微粒体中参与胡椒碱代谢的酶主要有CYP3A4和CYP2C9。     

Application of fuzzy-logic models for metabolic control analysis

A priori information or valuable qualitative knowledge can be incorporated explicitly to describe enzyme kinetics making use of fuzzy-logic models. Although restricted to linear relationships, it is shown that fuzzy-logic augmented models are not only able to capture non-linear features of enzyme kinetics but also allow the proper mathematical treatment of metabolic control analysis. The explicit incorporation of valuable qualitative knowledge is crucial, particularly when handling data estimated from in vivo kinetics studies, since this experimental information is scarce and usually contains measurement errors. Therefore, data-driven techniques, such as the one presented in this work, form a serious alternative to established kinetics approaches.     

Combining structural genomics and enzymology: completing the picture in metabolic pathways and enzyme active sites

An important goal of structural genomics is to complete the structural analysis of all the enzymes in metabolic pathways and to understand the structural similarities and differences. A preliminary glimpse of this type of analysis was achieved before structural genomics efforts with the glycolytic pathway and efforts are underway for many other pathways, including that of catecholamine metabolism. Structural enzymology necessitates a complete structural characterization, even for highly homologous proteins (greater than 80% sequence homology), as every active site has distinct structural features and it is these active site differences that distinguish one enzyme from another. Short cuts with homology modeling cannot be taken with our current knowledge base. Each enzyme structure in a pathway needs to be determined, including structures containing bound substrates, cofactors, products and transition state analogs, in order to obtain a complete structural and functional understanding of pathway-related enzymes.     

Flux control analysis for biphenyl metabolism by Rhodococcus pyridinovorans R04

The flux control coefficients of the four enzymes involved in the upper pathway of biphenyl degradation were determined from transient metabolite concentrations. The first enzyme was indicated as the major rate-limiting step of the pathway with a control coefficient of 0.48. The flux control coefficients of the other three enzymes were 0.03, 0.23 and 0.27, respectively. This is the first experimental evidence of the control step in the pathway of biphenyl degradation using metabolic control analysis.     

维生素C生物转化的代谢工程研究

本从代谢工程角度出发,综述维生素C(Vc)生物转化代谢研究进展。分别论述了3条产生Vc重要前体2-酮基-L-古龙酸(2-KLG)反应路线的代谢机制、反应酶系及代谢工程菌构建等方面的问题;并对Vc生物转化应用前景作了展望。     

Seasonal variations in the metabolic rates of zooplankton populations in a Thames Valley reservoir

Measurements of metabolic rates of natural populations of zooplankton were made using a closed bottle technique in situ. Results showed a marked seasonality inexplicable in terms of simple temperature functions. For this cladoceran dominated zooplankton, seasonal variations in metabolic response are attributed to a degree of temperature acclimation, to changes in the size structure and species composition of the populations and seasonal variations in food sources.     

Importance of various types of metabolic inhibition for cell damage caused by direct membrane damage

Cell damage is caused by energy depletion or by direct membrane damage, or a combination when a direct membrane damage affects energy depleted cells. In this report it was investigated whether the extent of direct membrane damage induced by lysophosphatidyl choline (LPC) or phospholipase C (PhC) on quiescent fibroblasts depended on the metabolic state of the cells. When glycolysis was inhibited cell damage was always extensively increased, whereas cell damage was also increased to a minor degree when exposed to PhC during sole inhibition of oxidative phosphorylation. Acceleration of glycolysis in cells with a low rate of glycolysis resulted in a dramatic improvement of the membrane susceptibility within a few minutes. Thus, susceptibility of the cell membrane to direct membrane damage depends on the metabolic state. The results also emphasize previous findings that glycolysis has a special role in maintaining membrane function and integrity.     

Quantitative approaches to the analysis of the control and regulation of microbial metabolism

Recently, a number of novel ways of considering the control, regulation and thermodynamics of microbial physiology have been developed and applied. We here present an overview of the new concepts involved, of their limitations and of the most recent attempts to deal with those limitations. We conclude that there no longer exist reasons of principle for vagueness in discussions of the control of microbial physiology and energetics. Further, the novel conceptual methods serve to remove part of the discordance between holistic and reductionistic views of microbial physiology.     

Metabolic control and compartmentation in single living cells

Microspectrofluorometry of cell coenzymes (NAD(P)H, flavins) in conjunction with sequential microinjections into the same cell of metabolites and modifiers, reveals aspects of the regulatory mechanisms of transient redox changes of mitochondrial and extramitochondrial nicotinamide adenine dinucleotides. The injection of ADP in the course of an NAD(P)H transient produced by glycolytic (e.g. glucose 6-phosphate, G6P) or mitochondrial (e.g. malate) substrate leads to sharp reoxidation (state III, Chance and Williams, 1955), followed by a spontaneous state III to IV transition, and an ultimate return to original redox steady state. The response to ADP alone is biphasic, i.e. a small oxidation-reduction transient followed by a larger reverse transient. Similarities between responses to injected ATP and ADP suggest possible intracellular interconversions. Sequential injections of glycolytic and Krebs cycle substrates into the same cell, produce a two-step NAD(P) response, possibly revealing the intracellular compartmentation of this coenzyme. A two-step NAD(P)H response to sequentially injected fructose 1,6-diphosphate and G6P indicates the dynamic or even structural compartmentation of glycolytic phosphate esters in separate intracellular pools. The intracellular regulation and compartmentation of bioenergetic pathways and cell-to-cell metabolic inhomogeneities provide the basis on which the quantitative biochemistry of the intact living cell may be reconciled with these in situ findings.     

Kinetic metabolic modelling for the control of plant cells cytoplasmic phosphate

A previously developed kinetic metabolic model for plant metabolism was used in a context of identification and control of intracellular phosphate (Pi) dynamics. Experimental data from batch flask cultures of Eschscholtiza californica cells was used to calibrate the model parameters for the slow dynamics (growth, nutrition, anabolic pathways, etc.). Perturbation experiments were performed using a perfusion small-scale bioreactor monitored by in vivo³¹P NMR. Parameter identification for Pi metabolism was done by measuring the cells dynamic response to different inputs for extracellular Pi (two pulse-response experiments and a step-response experiment). The calibrated model can describe Pi translocation between the cellular pools (vacuole and cytoplasm). The effect of intracellular Pi management on ATP/ADP and phosphomonoesters concentrations is also described by the model. The calibrated model is then used to develop a control strategy on the cytoplasmic Pi pool. From the identification of the systems dynamics, a proportional-integral controller was designed and tuned. The closed-loop control was implemented in the small-scale NMR bioreactor and experimental results were in accordance with model predictions. Thus, the calibrated model is able to predict cellular behaviour for phosphate metabolism and it was demonstrated that it is possible to control the intracellular level of cytoplasmic Pi in plant cells.