EI of the Phosphotransferase System of Escherichia coli: Mathematical Modeling Approach to Analysis of Its Kinetic Properties.

The mathematical model of the operation of the first enzyme of the Escherichia coli phosphotransferase system, EI, is proposed. Parameters of the kinetic model describing the operation of EI under different conditions are identified on the basis of a large amount of known experimental data. The verified model is employed to predict modes of operation of EI under both in vivo physiological conditions and in vitro nonphysiological conditions. The model predicts that under in vivo physiological conditions, the rate of phosphotransfer from EI to the second protein of the phosphotransferase system HPr by the dimer is much higher than by the monomer. A hypothesis is proposed on the basis of calculations that the transfer by a monomer plays a role in the regulation of chemotaxis. At submicromolar pyruvate concentration, the model predicts nonmonotonic dependence of the phosphotransfer rate on the substrate (PEP) concentration.

[A kinetic model of beta-galactosidase of Escherichia coli].

On the basis of previously published experimental data, the catalytic cycle of Escherichia coli galactosidase has been reconstructed. The enzyme plays a key role in the transcription regulation of lac operon. Using the proposed scheme, we have built a kinetic model that takes into account the reactions of hydrolysis, isomerization as well as galactosylation. Using the experimental data and optimization algorithms, all parameters of the model have been estimated. On the basis of the model, the features of beta-galactosidase functioning have been predicted in a wide range of conditions.

[A kinetic model of functioning and regulation of Escherichia coli isocitrate dehydrogenase].

A Rapid Equilibrium Random Bi Ter mechanism of formation of two dead-end complexes was proposed to describe the experimental data on the functioning of E. coli isocitrate dehydrogenase (IDH). A kinetic model for the enzyme functioning was constructed, which assumes that it is regulated through reversible phosphorylation by its kinase/phosphatase, which in turn is regulated by IDH substrates and central metabolites such as pyruvate (Pyr), 3-phosphoglycerate (3-PG), and AMP. It was shown using the model that increasing the concentration of these effectors results in an increase of the active part of IDH, thus leading to an increase in the Krebs cycle flux. We predict that the ratio of the phosphorylated and free forms of IDH (IDHP/IDH) is more sensitive to AMP, NADPH, and isocitrate concentrations than to Pyr and 3-PG. The model allows a realistic prediction of changes in the IDHP/IDH ratio, which would occur under changes of biosynthetic and energetic loading of the E. coli cell.

[A kinetic model of the cytochrome bf complex. Evaluation of kinetic parameters]. / Kineticheskaia model' tsitokhromnogo bf-kompleksa. Otsenka kineticheskikh parametrov.

A kinetic model of the cytochrome bf complex was developed on the assumption that the Q-cycle operates. The bf complex was considered as a membrane enzyme catalyzing the electron transfer from plastoquinol to plastocyanine, which is coupled with proton translocation from the chloroplast stroma to the thylakoid lumen. The dependence of the electron transfer rates on the value of the transmembrane electric potential was taken into account. The model was applied to describe the experimental data on the flash-induced turnover of cytochromes b, plastocyanine, and the kinetics of proton deposition in the thylakoid lumen. The estimation of model parameters was performed.

Signal processing at the Ras circuit: what shapes Ras activation patterns?

A systems biology approach is applied to gain a quantitative understanding of the integration of signalling by the small GTPase Ras. The Ras protein acts as a critical switch in response to signals that determine the cell's fate. In unstimulated cells, Ras switching between an inactive GDP-binding and active GTP-binding state is controlled by the intrinsic catalytic activities of Ras. The calculated high sensitivity of the basal Ras-GTP fraction to changes in the rate constant of GTP-hydrolysis by Ras can account for the carcinogenic potential of Ras mutants with decreased GTPase activities. Extracelluar stimuli initiate Ras interactions with GDP/GTP exchange factors such as SOS, and GTP-hydrolysis activating proteins such as RasGAP. Our data on freshly isolated hepatocytes stimulated with epidermal growth factor (EGF) show transient SOS activation and sustained Ras-GTP patterns. We demonstrate that these dose-response data can only be explained by transient RasGAP activitation, and not by merely switching off the SOS signal, e.g. by inhibitory phosphorylation of SOS. A transient RasGAP activity can be brought about by a number of mechanisms. A comprehensive kinetic model of the EGF receptor (EGFR) network was developed to explore feasible molecular scenarios, including the receptor-mediated recruitment of SOS and RasGAP to the plasma membrane, phosphorylation of RasGAP and p190 RhoGAP by soluble tyrosine kinases, and RasGAP interactions with phosphoinositides and p190 RhoGAP. We show that a transient RasGAP association with EGFR followed by the capture of RasGAP through the formation of complexes with p190 RhoGAP can account for data on hepatocytes. In summary, our results demonstrate that a combination of experimental monitoring and integrated dynamic analysis is capable of dissecting regulatory mechanisms that govern cellular signal transduction.

Kinetic model of imidazologlycerol-phosphate synthetase from Escherichia coli.

Based on the available experimental data, we developed a kinetic model of the catalytic cycle of imidazologlycerol-phosphate synthetase from Escherichia coli accounting for the synthetase and glutaminase activities of the enzyme. The rate equations describing synthetase and glutaminase activities of imidazologlycerol-phosphate synthetase were derived from this catalytic cycle. Using the literature data, we evaluated all kinetic parameters of the rate equations characterizing individually synthetase and glutaminase activities as well as the contribution of each activity depending on concentration of the substrates, products, and effectors. As shown, in the presence of 5 -phosphoribosylformimino-5-aminoimidazolo-4-carboxamideribonucleotide (ProFAR) and imidazologlycerol phosphate (IGP) glutaminase activity dominates over synthetase activity at sufficiently low concentrations of 5 -phosphoribulosylformimino-5-aminoimidazolo-4-carboxamideribonucleotide (PRFAR). Increased PRFAR concentrations resulted in decreased contribution of glutaminase activity and, consequently, increased the contribution of synthetase activity in the enzyme functioning.

[Kinetic model of primary processes of photosynthesis in chloroplasts. Fast phase of chlorophyll fluorescence induction under light of various intensity]. / Kineticheskaia model' pervichnykh protsessov fotosinteza v khloroplastakh. Opisanie bystroi fazy induktsii fluorestsentsii khlorofilla pri razlichnoi intensivnosti sveta.

A kinetic model was developed, which describing the system of generation and consumption of the transmembrane electrochemical proton potential delta mu H+ in primary photosynthetic processes. The model describes the catalytic cycles of photosystems I and II and the cytochrome b/f complex, as well as the ATP synthesis and passive leakage of H+, K+ [symbol: see text] Cl- ions through the thylakoid membrane. The dependence of the electron transfer rates on the value of transmembrane electric potential was taken into account. The model was applied to describe the experimental data on the registration of the fast phase of fluorescence induction. The model gives a realistic description of the fast phase of induction curves at different light intensities (from high to low).

[Kinetic modeling of energy metabolism and generation of active forms of oxygen in hepatocyte mitochondria]. / Kineticheskoe modelirovaniie énergeticheskogo metabolizma i generatsii aktivnyhkh form kisloroda v mitokhondriiakh gepatotsita.

Direct nonenzymatic oxidation of semiquinone by oxygen is one of the main sources of superoxide radicals (O2.-) in mitochondria. By using all the known data on hepatocyte mitochondria, we have revealed the correlation between the rate of superoxide generation by the bc1 complex and the transmembrane potential (delta psi). If the main electrogenic stage of the Q cycle is suggested to be the electron transfer between the cytochrome b hemes, then the rate of superoxide generation sharply increases when delta psi grows from 150 mV to 180 mV. However, this interrelation is ambiguous. Indeed, the increase of the generation rate with the growth of the potential can occur faster when succinate dehydrogenase is inhibited by malonate than when external ADP is exhausted. When the potential is changed by adding phosphate or potassium (K+), the rate of O2.- production remains constant, although the comparison of the rate values at the same delta psi reveals the effect of phosphate or potassium. It turned out that the rate of O2.- generation is a function of delta mu H rather than any of its components. Phosphate and K+ have practically no influence on delta mu H, since the change in delta psi is compensated by delta pH. The rate of superoxide generation by the bc1 complex is a multiple function of the electron-transfer activity of enzymes, the processes determining the membrane potential (e.g., loading), and of the oxygen concentration. The kinetic model proposed in this work may serve a tool to understand how the superoxide production is regulated.

[Regulation levels of photosynthesis processes]. / Urovni reguliatsii protsessov fotosinteza.

The basic mechanisms of kinetic regulation of photosynthetic processes are considered, which provide a strict light regulation of electron transfer in photosynthetic reaction centers and a more flexible regulation at the level of interaction of photosystems, transmembrane ion fluxes and coupling with dark reactions of the Calvin cycle. A generalized model was developed, which integrates the modern knowledge about photosynthetic processes of higher plants. The general principles of multilevel regulation in photosynthetic systems are discussed.

Quantification of short term signaling by the epidermal growth factor receptor.

During the past decade, our knowledge of molecular mechanisms involved in growth factor signaling has proliferated almost explosively. However, the kinetics and control of information transfer through signaling networks remain poorly understood. This paper combines experimental kinetic analysis and computational modeling of the short term pattern of cellular responses to epidermal growth factor (EGF) in isolated hepatocytes. The experimental data show transient tyrosine phosphorylation of the EGF receptor (EGFR) and transient or sustained response patterns in multiple signaling proteins targeted by EGFR. Transient responses exhibit pronounced maxima, reached within 15-30 s of EGF stimulation and followed by a decline to relatively low (quasi-steady-state) levels. In contrast to earlier suggestions, we demonstrate that the experimentally observed transients can be accounted for without requiring receptor-mediated activation of specific tyrosine phosphatases, following EGF stimulation. The kinetic model predicts how the cellular response is controlled by the relative levels and activity states of signaling proteins and under what conditions activation patterns are transient or sustained. EGFR signaling patterns appear to be robust with respect to variations in many elemental rate constants within the range of experimentally measured values. On the other hand, we specify which changes in the kinetic scheme, rate constants, and total amounts of molecular factors involved are incompatible with the experimentally observed kinetics of signal transfer. Quantitation of signaling network responses to growth factors allows us to assess how cells process information controlling their growth and differentiation.

Induction of nonselective permeability of the inner membrane in deenergized mitochondria.

Induction of the nonselective cyclosporin-sensitive pore in the inner mitochondrial membrane under conditions of complete dissipation of ion gradients and transmembrane potential was studied. This approach allows the kinetics of Ca2+-dependent pore opening and the preceding processes of induction to be studied separately. The effects of mitochondrial heterogeneity were also minimized. We found that the kinetics of pore opening can be described by a minimal two-step scheme where only the rate constant at the first step depends on Ca2+ concentration. Oxidation of pyridine nucleotides in the matrix caused a slow transition in the pore complex and decreased the apparent dissociation constant of the Ca2+-binding site from >1 mM to approximately 30 microM. N-Ethylmaleimide (but not disulfide-reducing agents) prevented and slowly reverted the pore induction process. Data suggesting allosteric modulation of the pore by pyridine nucleotides are presented.

A model of O2.-generation in the complex III of the electron transport chain.

Oxidation of semiquinone by O2 in the Q cycle is known to be one of the sources of superoxide anion (O2.-) in aerobic cells. In this paper, such a phenomenon was analyzed using the chemical kinetics model of electron transfer from succinate to cytochrome c, including coenzyme Q, the complex III non-heme iron protein FeSIII and cytochromes bl, bh and cl. Electron transfers from QH2 to FeSIII and cytochrome bl were assumed to occur according to direct transfer mechanism (dynamic channelling) involving the formation of FeS(red)III-Q.- and Q.--cytochrome bl complexes. For oxidation/reduction reactions involving cytochromes bh and bl, the dependence of the equilibrium and elementary rate constants on the membrane potential (deltapsi) was taken into consideration. The rate of O2.- generation was found to increase dramatically with increase in deltapsi above the values found in State 3. On the other hand, the rate of cytochrome c reduction decreased sharply at the same values of the membrane potential. This explains experimental data that the O2.- generation at State 4 appears to be very much faster than at State 3. A mild uncoupling in State 4 can markedly decrease the superoxide generation due to a decrease in deltapsi below the above mentioned critical level. DeltapH appears to be equally effective as deltapsi in stimulation of superoxide production which depends, in fact, upon the deltamuH+ level.

'Channelled' pathways can be more sensitive to specific regulatory signals.

In 'simple' metabolic pathways the response to an external signal is readily described in terms of the effect of the signal on its receptor enzyme and the control exerted by that enzyme. We show here that in the response of 'channelled' pathways to such a signal, additional terms appear that reflect the direct enzyme-enzyme interactions. They tend to enhance the responsiveness of the pathway. The normalized value of the response is called the signal transduction coefficient. We show that in channelled pathways these coefficients are usually larger than in corresponding non-channelled (simple) pathways.