Comparison of control analysis data using different approaches: modelling and experiments with muscle extract

Abstract

Experimental and model studies have been performed to characterize the control properties of hexokinase and phosphofructokinase in muscle glycolysis and to examine the nature of error associated with experimental flux control coefficient determinations. Different approaches of metabolic control analysis, classical titration, co-response analysis and kinetic modelling indicated that flux control coefficients could be reliably estimated experimentally for the upper part of glycolysis. The kinetic parameters applied to construct the mathematical model were determined in muscle extract under similar conditions used for flux studies. If the kinetic parameters of commercial enzymes are introduced into the model the control analysis data cannot be trusted. Co-response analysis can also be successfully applied to determination of the flux control coefficients of the system. However, the involvement of a rapid-equilibrium enzyme, such as glucose 6-phosphate isomerase, could result in estimation errors for the relevant co-response coefficients that are propagated into the elasticity matrix. If the co-response coefficients related to isomerase activity are replaced by the values obtained by kinetic modelling, the values of elasticities are correct. Our data also suggest that in the upper part of glycolysis hexokinase mainly controls the pathway flux whereas phosphofructokinase exerts dominant control on the turnover of internal metabolite stocks inside the system.

Flux control and excess capacity in the enzymes of glycolysis and their relationship to flight metabolism inDrosophila melanogaster

“…It has also been speculated that this association is required to maintain tight flux regulation. Numerous studies have implicated HEX and GLYP as regulatory enzymes in glycolysis (30)(31)(32). Our results in D. melanogaster show that as HEX-A and GLYP activities continue to be reduced, capacities are exceeded, regulation is disrupted, and flux is reduced.…”

Isozymes of plant hexokinase: Occurrence, properties and functions

“…In animals, HK has been shown to exert a high level of control over glycolytic flux. Thus, values of flux control coefficients between 0.7 and 1 have been measured in mammalian erythrocytes, liver, heart, insulinoma and muscle cells (Rapoport et al, 1974;Torres et al, 1988;Kashiwaya et al, 1994;Wang and Iynedjian, 1997;Puigjaner et al, 1997;Jannaschk et al, 1999). In plants, however, the control coefficient of HK over glycolytic flux has not been assessed.…”

Effects of Cadmium and Mercury on the Upper Part of Skeletal Muscle Glycolysis in Mice

“…C J E  = d ln (flux)/d ln (enzyme activity) . In previous works, we observed that, in extracts of mouse skeletal muscle, hexokinase (HK) and phosphofructokinase (PFK) shared control of the metabolic flux in the upper part of glycolysis [15], [25]. Also, we characterized the irreversible inhibitory effects of pre-incubation with copper and we identified HK and PFK as the main targets of copper [15].…”

“…The final concentrations in the cuvette were 2 mM NADH, 2 mM MgATP, 10 mM Glc, 20 mM PC, 3 U/ml CK, 3.5 U/ml TPI and 0.5 U/ml GDH. NADH consumption was monitored at 385 nm (ε 385 nm NADH  = 0.75 mM −1 cm −1 ), as described by Puigjaner et al [25], using a Shimadzu UV-2101PC Spectrophotometer with 1-cm light path cells. These assay conditions are not quite representative for mouse muscle in vivo , but in view of the considerable amount of consensus building required to achieve proper in vivo standard conditions [27], we here reverted to conditions that are not far off from the in vivo state and that our previous work [15], [25] has shown to work well.…”

“…Two detailed rate- plus balance-equation models were developed based on our previously published paper [25]. Network and equations are described in Fig.…”