THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1992 by The American Societyfor Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 5, Issue of February 15,pp. 31069114,1992 Printed in U.S.A.

Systems Analysis of the Tricarboxylic Acid Cyclein Dictyostelium discoideum

11. CONTROLANALYSIS*
(Received for publication, April 16, 1991)

Kathy R. Albe and Barbara E.Wright

From the Diuision of Biological Sciences,University of Montana, Missoula, Montana 59812

A steady-state computer model of the tricarboxylic cycle in Dictyostelium diecoideum was analyzed using metabolic control theory. The steady state had variations of less than 0.04% over thelast half of the simulation for both metabolite concentrations and fluxes. Metabolite and flux control coefficients were determined by varying enzymatic activities within 2% of their initial values and simulating the responses of metabolite concentrations and fluxes to these changes. Under these conditions, summation properties were met for most metabolite and all flux control coefficients. Maximum flux control coefficients were found for succinate dehydrogenase (0.35), malic enzyme (0.24), and malate dehydrogenase (-0.18). Comparable control was found for the reaction supplying pyruvate (0.14) and for the sum of the input amino acids (0.43), which serve as an energy source for D. discoideum. The time-dependent processes bywhich a new steady state wasestablished were examined after increasing malic enzymeor malate dehydrogenase activities. This provided a method for an analysis of the mechanisms by which the observed control coefficients were generated. In addition, the effects of increasing the stimuli within 5-20% of the original enzyme activity were examined. Under these conditions, more typical of experimental stimuli and measurable responses, the metabolic model failed to return to steady state, and thus summation properties were not met. Whether true steady states everoccur or whether metabolic control theory can be applied in vivois discussed.

acid model developed in our laboratory (see companion paper). The data used in thismodel were collected from Dictyostelium discoideum and included both flux relationships based on an extensive analysis of tricarboxylic acid cycle flux using radioactive tracers and the characteristics of enzymes isolated from the same organism and analyzed using standard i n uitro techniques. After changing enzymatic activities, the responses of metabolite concentrations and fluxes occurring over time were determined using a computer simulating program called METASIM. METASIM models, constructed using these experimentally determined data, have not only reproduced a single data set butalso have predicted the organisms i n uiuo responses to perturbations by exogenous metabolites and to mutations in the pathways (see companion paper). This analysis of the tricarboxylic acid modelshould not only enable us to examine theoretical questions about MCT but also to predict the i n uiuo distribution of control. Control analysis defines two control coefficients that quantify the response of metabolite concentration or pathway flux to changes in enzymatic activity of one of the enzymes in the pathway (2-5). The mathematical descriptions of these coefficients are as follows.
CM--=-

v, - 6 Vi/ V,

61nM 61n Vi (metabolite control coefficient)

CJ v; --=- 6 Vi/Vi

6ln V,

Many theoretical and experimental techniques have been used to examine the control and regulation ofmetabolic pathways in living cells. Recently, systematic approaches have been developed for the analysis of complex metabolic networks. Metabolic control theory (1-5), flux-oriented theory (6), and biochemical systems theory (7) all propose a mathematical analysis of metabolic pathways. Mathematically, they can be interrelated (8); differences occur in terminology, emphases, and mathematical generality. Because of its apparent simplicity in description and application and its use of familiar biochemical terminology, metabolic control theory (MCT) has been the most widely applied. For these reasons, MCT has been used to analyze a steady-state tricarboxylic

These control coefficients are normalized, and thustwo summation properties can be defined (3-5). Simply stated, the sum of the normalized effects of individually changing all enzyme activities ( V , i = 1,2,. . .n)on any metabolite concentration ( M ) is 0, and on pathway flux ( J ) is 1, i.e.
i-1

2 cc=o

i= 1

c CJ, = 1

* This work was supported by National Institutes of Health Public Health Service Grant AG03884. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisernent in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. The abbreviation used is: MCT, metabolic control theory.

In addition to control coefficients, MCT defines other relationships, called elasticities, derived from the properties of the isolated pathway enzymes. Connectivity properties describe the relationship of elasticities to systemic control coefficients (3). Connectivity and summation properties are necessary for the calculation of control coefficients in experimental systems in which it is impossible either to change all enzymatic activities independently or to measure all responses of the system. MCT states that this analysis is applicable to any experimental or theoretical system that has a stable steady state and in which enzymatic activities can be changed and systemic responses measured. To increase enzymatic activity,

3106

Control Analysis of Tricarboxylic Acid Cycle in D.discoideum

experimental biologists have added enzymes to crude extracts (9, IO), analyzed mutants with increased activity (11),and used variably expressible plasmids (12-14). However, preparation of crude extracts destroys both microcompartmentation and protein interactions which are known to occur in uiuo (15-18).As there is a very poor correlation between enzymatic activity in uitro and in uiuo, such experiments are unlikely to provide information about the regulation of the pathway i n vivo (19). To decrease enzymatic activity, investigators have used inhibitors (12, 20-24), mutants with decreased enzymatic activity (25, 26), and variably expressible plasmids (14). Of necessity, most of these techniques result in large changes in enzymatic activity. When enzymatic activity at a step in a pathway is greatly increased, response of pathway flux to that enzyme decreases; that is, the enzyme becomes less rate controlling, and its flux control coefficient decreases. It is also true that as enzymatic activity is decreased at a particular step, that enzyme becomes more rate controlling, and itsflux control coefficient increases. Our analysis of the tricarboxylic acid cycle is compatible with these observations; that is, when only decreased enzymatic activity was used in the calculation of flux control coefficients, each coefficient was overestimated, and thus the sums were greater than one (see Table 5 ) . This observation is generally not made or discussed in papers dealing with experimentally analyzed pathways, as only a few coefficients are measurable, and the remaining coefficients are calculated from connectivity and summation properties (3, 27). This would imply that flux control coefficients determined in these analyses are always over- or underestimated. Although these phenomena are generally not acknowledged in experiments designed to measure control, these issues have been discussed (14, 26, 28). Experimental measurements of control also depend upon the in uitro measurement of enzymatic activity. This procedure cannot accurately measure in uiuo enzymatic activity, as intracellular organization is disrupted, inhibitors or activators are lost, and the measurements of activity are generally done under ideal, rather than physiological conditions. The use of relative enzymatic activities also has serious experimental problems such as differences in both extraction and in activation of pathway enzymes. What may be ideal assay conditions for the referent enzyme may not be for other pathway enzymes and will generally differ from physiological conditions. Although such analyses claim to quantify control, based on the above inherent problems in experimental measurements, they can at best be a relative and qualitative description of the distribution of control in the native pathway, regardless of the quantities assigned to particular steps in the process. Control analysis has also been applied to theoretical constructs of linear and branched pathways, cycles, loops, and moiety-conserved substrates (29-33). These types of analyses predict the distribution of control, i.e. where and how control would be distributed. Paper experiments have also been constructed to examine the molecular basis of dominance (34, 35). These typesof analyses may give insight into thegeneral structure of control distribution in pathways although they cannot give specific information about controlof a particular pathway. As the METASIM model is a mathematicaldescription of the tricarboxylic acid cycle consisting of independent equations which define individually catalyzed fluxes (see companionpaper) an analysis of control using MCTcan be performed. To elucidate the problems in applying MCT to experimental systems andto examine model constraints, changes were made in thespecified enzymatic activities (Vvivo;

3107

see companion paper) of each enzyme in the pathway and the responses of the model simulated.
METHODS~

RESULTS

There are three major advantages to using computer models for MCT analysis: 1)much smaller changes can be made and smaller responses measured; 2) all enzymatic activities can be changed independently; 3) all system responses can be measured. Control analysis is based on differential changes, thus the smaller the changes, the closer to theory. MCT also requires a stable steady state, which can only be determined if all variables can be measured. The structure of the mitochondrial model used for these analyses is shown in Fig. 2. The companion paper discusses the development and use of this model. In the unperturbed, steady-state model input amino acid equaled output COP,and the variation between 5 and 10 min of simulation was 0.02% or less for metabolite concentrations and 0.04% or less for fluxes. This variation occurred in the fourth place of the values reported by the METASIM simulation and was probably the result of computer round-off error in the calculation of metabolite concentration and fluxes rather than inherent instability of the steady state. Thus,for purposes of this analysis, the model wasin steadystate. When 1 or 2% changes in enzyme activities were made, most simulated values returned to new steady-state values, i.e. less than 1% variation between values at 5 and 10 min of the simulation. However, several responses had a variation of 1%or greater for some of the changes in enzymatic activities (data not shown). Because the control analysis was performed 10 min after the application of the stimulus, these more unstable metabolite concentrations, and flux responses were examined by comparing the values at 9 and 10 min to determine the extent to which a new steady state had been reached (Tables 1 and 2). Fewer time-dependent variations were seen in flux values than in metabolite concentrations. The most unstable metabolite concentration was pyruvate, which showed maximal concentration variations in all the simulations examined. Because it was unlikely that pyruvate or acetyl-coA concentrations had obtained new steady-state values after 10 min of stimulation, the control coefficients calculated for these metabolites would not quantitate control accurately. Metabolite control Coefficientsand flux control coefficients are shown in Tables 3 and 4, respectively. These coefficients were determined from changes in stimuli of 1 or 2% above and below the original steady-state value for enzymatic activities. As noted previously, pyruvate and acetyl-coA concentrations did not return to new steady-state values. This is reflected by the deviation from the theoretical summation of 0, -0.5, and -0.19, respectively (see Table 3).The summation of 2-ketoglutarates metabolite control coefficients also showed a deviation from theory (0.13 uersus 0), but the basis for this variant behavior was not obvious from the observed percent variationin metabolite concentration (Table1).However, the concentration of 2-ketoglutarate is considerably lower than pyruvate (0.01 uersus 0.3 mM), and smaller variations from steady state may be more significant to its calculated metabolite control coefficients. These metabolite control coefficients should qualitatively reflect the distribution of

* Methods,Figs. 1 and 3, and Tables 1, 2, 4, and 8 are presented in miniprint at the end of this paper. Miniprint is easily read with the aidof a standardmagnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

3 108

Control Analysis of TricarboxylicAcid Cycle in D. discoideum

FIG. 2. Steady-state model of the tricarboxylic acid cycle in D. diecoideum. The encircled reaction numbers are catalyzed by enzymes, which were isolated from D. discoideum and kinetically characterized. All metabolite pools are intramitochondrial as defined by Kelly et al. (43,44). Abbreviations are as shown in the legend to Table 3.

TABLE I11 Control coefficients (C,'") of metabolite responseto changes in enzymatic activities Control coefficients were calculated from metabolite concentrations determined after 10-min simulations in which enzyme activities were changed ? 2% of the original steady-state value (see "Results"). The procedure for the calculation of the control coefficient is described in Figure 1. Glu DH, glutamate dehydrogenase; 2-KGDC, 2ketoglutarate dehydrogenase complex; SDH, succinate dehydrogenase; MDH, malate dehydrogenase; ME, malic enzyme; PDC, pyruvate dehydrogenase complex; CS, citrate synthase; Isoc DH, isocitrate dehydrogenase; AspTA, aspartate transaminase; AlaTA, alanine transaminase; DH, dehydrogenase; PROT, protein; OAA, oxaloacetate; ACO, acetyl-coA; Isoc, isocitrate; Pyr, pyruvate; Glu, glutamate; Asp, aspartate; Ala, alanine; CIT, citrate; 2KG, 2-ketoglutarate; SUC, succinate; FUM, fumarate; MAL, malate.
Stimulus"
OAAl ACO OAA2

Isoc

Pyr

Glu

Asp
C,M*

Ala

CITl

2KG1 FUMl SUCl

ma11

1. Glu DH 0.020 0.020 -0.070 2. 2-KGDC -0.040 -0.028 0.21 3. SDH 0.45 0.97 -1.96 4. Fumarase 0 0.008 -0.012 0.003 5. MDH 0.16 0.72 -4.30 -0.21 -0.98 5.86 6. ME 7. Ala + Pyr 0.088 0.56 0.028 8. PDC 0 0.99 -0.11 0.040 -0.072 -0.010 0.43 -0.12 1.07 -0.020 0.12 0.36 0.020 -0.019 -0.007 9. OAA2 + Asp 10. Asp + OAA2 -0.040 0.072 -0.43 11. cs -1.59 0.015 -0.63 -1.37 -0.048 12. CITl * Isoc 0 0 -0.005 13. Isoc DH 0 0 -1.03 -0.005 14. Glu + SUCl 0.080 0.082 0.48 0.063 -0.30 0.060 -0.16 -0.14 -0.10 -0.59 0.39 0.12 15. AspTA 0.040 0.048 -0.37 16. AlaTA 0.008 0.068 -0.53 17. OAAl + OAA2 -0.94 0.092 -0.56 18. Asp + OAAl 0.048 0.068 -0.38 19. SUCl + Glu -0.080 -0.12 0.30 20. OAAl +ASP -0.048 -0.068 0.38 21. PROT +Asp 0.056 0.22 -0.67 22. PROT-ACO 0.12 -0.052 1.69 23. PROT- SUCl 0.12 0.21 -0.71 24. PROT+ FUMl 0.040 0.088 -0.24 25. PROT + ALA 0.068 -0.028 1.07 26. PROT + GLU 0.080 0.14 -0.51

0.008 -0.003 0.52

-0.15 0.55 -3.35 -0.033 -0.003 0.011 0.16 -10.91 -0.22 14.80 0.065 -0.033 -0.36 -0.30 1.70 -0.020 0.003 -0.038 0.017 -0.17 0.011 0

-0.010 -0.17 -1.29 0.77 -1.04

-0.054 0.36 1.58 0.70 -0.94

-0.035 0.11 -0.62 -0.006 -1.15 1.56

0.008 0 0.50 0 0.16 -0.21 0.060 0

0.12 -0.77 -2.51 -0.020 -0.012 -0.50 -0.85 1.17 0.082 0.44 0.65 0

0.084 0.13 -2.79

-0.41 0.55 -0.004

0.018 0.018 0.88 -0.62 -0.23 0

-1.08 -0.37 0.12 -0.12 0.020 -0.020 0.019 -0.010 0 -0.98 -1.38 -1.41 -0.95 0.60 0.94 -1.43 3.99 -1.56 -0.48 3.08 -1.16 0.18 0.012 0.029 -0.024 0.020 -0.18 -0.54 0 0 0 0 0.15 -0.017 0.15 0.11 0.10 -0.11 0.18 -0.033 0.58 0.090 -0.017 0.59 -0.35 -0.016 -0.35 -0.34 0.14 0.34 0.58 -0.31 -0.15 0.016 -0.14 -0.070 -0.006 -0.004 -1.00 0

0.008

0 0

0.004 0 -0.10 0.039 -0.006 0.019 -0.48 -0.019 0.079 0.54 0.71 0.066 0.31 0.43

0 0 -0.007 0.008 -0.007 0.008 -0.082 -0.007 0.008 0.098 0.12 0.058 0.055 0.068

0.003 0.003 0.013 0.010 -0.061 -0.010 0.10 0.10 0.085 0.043 0.065 0.063

-0.076 0 -0.19 0.053 0.068 0 -0.15 0.020 -0.051 0.020 -0.11 0 0.15 -0.060 -0.38 0 0.10 -0.020 -0.12 0.096 0.052 0.46 0.092 0.81 0.68 -0.089 0.094 -0.045 0.040 0.076 0.060 0.48 0.61 0.44 -0.006 0.060

0.014 0.009 0.80 0 -0.86 -0.39 0.077 0 -0.009 0.009 0.014 0 0 0.077 0 0.009 -0.014 -0.009 -0.077 -0.009 0 0.091 0.12 0.054 0.054 0.068

Sum

0.012 -0.079 -0.021 0.094 -0.503 -0.001 -0.192 0.044 0 0.046 0.063 0.089 0.126 Stimulus, enzyme changed in activity; numbers refer to reactions in Fig. 2. CIM, Metabolite control coefficient calculated from the response, M, to the stimulus, i, where M is the metabolite concentration response and i is the enzyme changed in activity.

Control Analysis of Tricarboxylic AcidCycle in D. discoideum

control even for metabolite concentrations which failed to establish new steady-state values over the time of the simulation. The effect of each enzyme variation on the individual fluxes is shown in Table4; the theoretical summationof 1.0 is found in all cases. Obviously,this information is important toassess control for each individual enzyme flux, but it gives little information aboutthe effect of changing particular enzymatic activities on the pathway flux. By defining pathway flux as the production of COz,either by reactions within the tricarboxylic acid cycle, or by total COz generation, a composite flux control coefficient can be calculated (Table 5). From these coefficients, malate dehydrogenase, succinate dehydrogenase, and malic enzyme share the majority of control over tricarboxylic acid cycle flux for enzymes within the cycle. The supply of two-carbon units is important in maintaining cycle flux, thus input from alanine to pyruvate (reaction 7) and from protein degradation to alanine (reaction 25) had significant flux control ( C A+ ~ pyr ~ = 0.14; Cpmt ~1~ = 0.25). Summation propertieswere met for these composite values as well as for the individual fluxes, indicating that the observed variations in fluxes or concentrations from steady state (Tables 1 and 2) were not significant with respect to this analysis. Another observation, consistent with obtaining the

3 109

theoreticalsummation values, is that opposite and equal reactions (9 and 10, 14 and 19, 18 and 20) have opposite and equal flux control coefficients (Tables 4 and 5). Selected metabolite control coefficients (Table 6 ) and all the Con flux control coefficients (Table 7) were calculated after changes in enzymatic activity of 5-20%. These control coefficients did not meet summation properties. These differences between the summation of the calculated control coefficients and theory probably result from the failure of the simulations to establish a new steady state after these relatively large perturbationsin enzymatic activities. Obvious deviations between flux control coefficients calculated from small perturbations versus those calculated from larger perturbation are those reported for malate dehydrogenase and malic enzyme. If these enzymatic activities were decreased 5-20%, then positive flux control coefficients were observed. If these enzymatic activities were increased 5-20%, then negative flux control coefficients were observed (see TimeEvents and Mechanisms in the Re-establishment of Steady-state in the Miniprint).
DISCUSSION

TABLE V Effect of changing enzymaticactivity on COS production There is no net COS production when glutamate is converted to succinate since succinate is converted to glutamate at an equal rate (reaction 14 = reaction 19). Flux control coefficients were calculated from fluxes determined after 10-minute simulations in which enzyme activities were changed +2% of the original, steady-state value (see Results). The procedure for the calculation of control coefficients is described in Figure 1. Abbreviations are as in Table 3.
Stimulus
C:eYceCo~b

C:co2c

0.027 0.020 0.063 0.043 0.35 0.45 0 0 5. -0.18 -0.15 6. 0.24 0.21 7. 0.14 0.11 8. 0 0 9. 0.009 0 Asp 10. -0.009 0 11. cs -0.011 0 12. CITl + Isoc 0 0 13. Isoc DH 0 0 Glu 14. + SUCl -0.023 0.007 15.AspTA -0.054 -0.037 16.AlaTA 0.007 0.011 17. OAAl + OAA2 -0.020 -0.011 18. Asp + OAAl -0.009 -0.003 19. SUCl + Glu 0.023 -0.007 20. OAAl + Asp 0.009 0.003 0.034 0.054 21. PROT 4Asp 0.19 0.089 22. PROT + ACO 23. PROT 4SUCl 0.031 0.057 0.018 0.028 24. PROT 4 FUMl 0.12 0.094 25. PROT + Ala 26. PROT + Glu +0.034 +0.041 Sum 0.993 1 . 0 0 6 .~. . a Stimulus: enzyme changed in activity; numbers referto reactions in Fig. 2. b c JeydeCq.. control coefficient calculated from the response, J cycle COS,to stimulus, i, where J cycle CO, is the sum of the responses of fluxes 2 and 13, and i is the enzyme changed in activity. c CJtOtalCO 2: control coefficient calculated from the response, J total COS,to stimulus, i, where J total CO, is defined as the sum of the responses of fluxes 2,6,8, and 13, and i is defined as enzyme changed in activity.
1. 2. 3. 4.

Glu DH 2-KGDC SDH Fumarase MDH ME Ala + Pyr PDC OAA2 4 Asp + OAA2

The application of MCT assumes the establishment of a new, stable steady state after changing enzymatic activities. The definition of steady state is that mass in equals mass out. However, experimental measurements of mass in and/or out maybe impossible. For the tricarboxylic acid cycle in D. discoideum, input is amino acid from protein degradation, and Since there are many other reactions in vivo output is Con. using amino acids as substrates and producing COz, neither mass in nor mass out is unique to thetricarboxylic acid cycle. Experimentally, it is therefore impossible to measure mass in or out for this model directly, and indirect methods must be employed (see companion paper). Thus, determination of steady state in vivo will depend upon the variables which can be measured, the sensitivity and specificity of the assays used, and upon the time at which the measurements are made. To illustrate the problem in choosing the variables to be measured, consider the METASIM model in which net mass in equaled net mass out before and after perturbation. If the stability of the steady state, after perturbation, were based on oxaloacetate concentrations, then all simulations resulted in stable steady states. However, if the stability of the same simulations were based on pyruvate concentrations, then none of the simulations returned to a stable steadystate. By analogy, in experimental systems in which mass in and/or out may not be measurable and only a selected number of metabolite concentrations can be determined, it would be nearly impossible to establish clearly the existence of a stablesteady state. In an explicit mathematical description of a pathway, such as this METASIM model, the stability of the steadystate depends on the precision of the estimation method, the number of significant figures reported for metabolite concentrations and fluxes, and the buffering and feedback present. In experimental systems, variationin values of 0.1% are difficult, if not impossible, to quantify but are reported by the METASIM model. Most metabolite concentrations and fluxes in the METASIM model did establish new steady-state values as demonstrated by obtaining theoretical values for summations properties. Although the model failed to return to steady state afterlarge perturbations, the in vivo system may establish a new quasi-steady state after such perturbations, as there is considerably more buffering of metabolite concentrations and more complexity of enzymatic reactions than can be usefully analyzed in the METASIM model.However, the inherent experimental difficulties both in changing parame-

3110

Control Analysis of Tricarboxylic Acid Cycle in D.discoideum

TABLEVI Selected metabolite control coefficients(Ci") using larger chnngesin enzymatic activity
Stimulus' Change stimulus in
(0.80-0.95)"

Change stimulus in

(1.05-1.30)b ASD
CITl

OAAl

Ism

ASD

CITlOAAl MALl

Isoc

MALl

1. GluDH 2. 2-KGDC

3. SDH 4. Fumarase 5. MDH 6. ME 7. Ala + Pyr 8. PDC 9.OAA2 +Asp 10. Asp + OAA2 11. cs 12. CITl + Isoc 13. Isoc DH 14. Glu + SUCl 15.AspTA 16.AlaTA 17. OAAl + OAA2 18. Asp -+ OAAl 19. SUCl+ Glu 20. OAAl + Asp 21. PROT + Asp 22. PROT + ACO 23. PROT + SUCl 24. PROT + FUMl 25. PROT + Ala 26. PROT + Glu Sum

0.021 -0.027 1.00 0.007 0.70 -3.19 0.014 -0.13 -0.069 0.068 -1.41 0.003 0 0.11 0.042 0.064 0.098 0.064 -0.11 -0.060 0.18 -0.10 0.17 0.059 -0.054 0.14

0.010 0 0.65 0.003 0.43 0.11 0.066 0.002 -0.013 0.020 0.041 0 -1.05 0.058 0.005 0.005 0.037 0.032 -0.052 -0.011 0.091 0.11 0.082 0.029 0.073 0.062

CzM -0.047 0.41 1.64

0.010 0.14 -1.53 -0.27 -0.048 0.34 -0.34 -0.65 0 0 -0.13 -0.32 -0.024 -0.41 -0.33 0.16 0.30 0.53 -0.30 -0.11 0.015 -0.15 -0.065

0.009 -0.002 0.63 0.004 0.41 0.11 0.064 0.002 -0.014 0.021 0.038 -1.00 0 0.056 0.003 0.005 0.037 0.033 -0.053 -0.012 0.087 0.10 0.078 0.028 0.070 0.058

0.012 0.010 0.95 0.003 -0.65 -0.22 0.078 0.002 -0.011 0.012 0.023 0 0 0.075 -0.005 0.011 -0.007 0.024 -0.072 -0.010 0 0.10 0.097 0.040 0.058 0.070

0.030 -0.026 0.79 0.005 1.95 -1.00 0.042 -0.093 -0.082 0.081 -1.34 0 0 0.13 0.052 0.065 0.094 0.072 -0.12 -0.072 0.26 -0.026 0.19 0.079 -0.024 0.16

0.013 -0.003 0.30 0.004 -0.026 -0.67 0.066 0 -0.024 0.014 0.020 0 -1.04 0.060 0 0.004 0.022 0.015 -0.066 -0.021 0.10 0.060 0.075 0.038 0.042 0.060

CZM -0.068 0.33 1.25 0.007 1.06 -0.014 -0.34 -0.031 0.41 -0.40 -0.49 -0.003 0 -0.18 -0.38 0 -0.33 -0.37 0.14 0.37 0.63 -0.24 -0.14 0.022 -0.12 -0.068

0.013 -0.003 0.28 0.002 -0.026 -0.65 0.061 0.002 -0.024 0.015 0.017 -1.00 0 0.057 0.002 0.002 0.017 0.014 -0.064 -0.020 0.10 0.056 0.074 0.035 0.041 0.057 -0.94

0.018 0.006 0.44 0.004 -0.95 -0.81 0.074 0 -0.014 0.011 0.019 0 0 0.080 -0.006 0.004 -0.009 0.013 -0.084 -0.015 -0.003 0.056 0.092 0.051 0.036 0.070 -0.92

-1.18 -2.41 0.79 0.76 1.04 0.69 -0.96 1.22 Enzyme activity is changed to (0.80,0.85,0.90, 0.93, or 0.95) X original steady-state value. *Enzyme activity is changed to (1.05, 1.07, 1.10, 1.15, 1.20, or 1.30) x original steady-state value. Stimulus: enzyme changed in activity; number refer to reactions in Fig. 2.

ters andmeasuring variables i n uiuo make the application and significance of MCT analysis questionable. Control analysis of realistic and predictive metabolic models may provide a useful tool for examining questions n uiuo. about applying MCT as well as for assessing control i In a metabolicmodel all parameters are explicitly stated; independent and specific changes in these parameters canbe made; and very small changes in parameters, both increasing and decreasing around the operating point, can be made and small responses measured. In Dictyostelium major control points within the tricarboxylic acid cycle were identified at reactions catalyzed by malate dehydrogenase, succinate dehydrogenase, and malic enzyme (Table 5 ) . Citrate synthase, commonly thought to be a rate-controlling enzyme in the tricarboxylic acid cycle, was found to exert minor control in this system. The rest of the control was distributed among the input fluxes from alanine to pyruvate and from protein : ) of the input from protein degradation. The sum (ZfZzl C was 0.43, with the majority from reaction 25 (CJ,Z5 = 0.25). The supply of C-2 fragments becomes importantin D. discoideum as more four- and five-carbon tricarboxylic acid cycle intermediates are formed from amino acid degradation 24 + 26) = 1.09) than two-carbon (reactions 21 + 23 25) = 0.91). To overcome this fragments (reactions (22 imbalance in precursors, an anaplerotic pathway (providing acetate to thecycle) is formed by malic enzymeand pyruvate dehydrogenase complex. Malate, as the branchpoint metabolite, can serve either as aprecursor for oxaloacetate or can be decarboxylatedto form pyruvate. Since malate dehydrogenase and malic enzyme are at this key branchpoint, it is reasonable to expect, although not obvious that control at this point would be a key feature in Dictyostelium.

Canela et al. (45)constructed a model of the tricarboxylic acid cycle in muscle and used MCT to analyze control. This modelwas primarily theoretical and lacked the extensive experimental data used in our model of the tricarboxylic acid cycle in D. discoideum. However, they also found that variations at their branchpoint metabolite, fumarate, resulted in changes in the distribution of control and that succinate dehydrogenase had major control (0.19-0.39), depending upon the conditions of analysis. Citrate synthase also had a large control coefficient, but there was no anaplerotic pathway for acetate synthesis as found in D.discoideum. Although Dictyostelium is an unusual organism, other organisms used as experimental systems dogive support for some of the results of our analysis. Succinate dehydrogenase was identified as a major control site in tricarboxylic acid cycle flux in rat brain damaged by cold-induced edema; in normal brain this was not the case (46). Succinate dehydrogenasemay also play a role in respiratory control in the mitochondrion since it is a linkage point between the tricarboxylic acid cycle and proton flux (23). The control exerted by citrate synthase was studied extensively using a variably expressible plasmid in Escherichia coli (14). Citrate synthase was a major control point if the bacterium were grown on acetate whereas it exerted only minor control if the bacterium were grown on glucose. This suggested that citrate synthase served as a branchpoint regulator when twocarbon units were supplied; basically the enzyme provided a shunt for acetate either to degradation via the tricarboxylic acid cycle or to lipid synthesis (14). Predicting Flux Response fromEnzymeParameters-If there were a simple way of predicting the relative size of the control coefficients from familiar enzyme parameters rather

Control Analysis of Tricarboxylic Acid

TABLE VI1 Flux control coefficients C () : using larger changes in enzymatic activity Flux control coefficients were calculated from fluxes determined after ten minute simulations, as previously described (see Figure 1). Abbreviations are as in Table 3.
Chanee in stimulus Chanee in stimulus

Cycle in D. discoideum

3111

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

Glu DH 2-KGDC SDH Fumarase MDH ME Ala + Pyr PDC OAA2+Asp Asp 3 OAA2

cs

CITl + Isoc Isoc DH Glu + SUCl AspTA AlaTA OAAl + OAA2 Asp + OAAl SUCl + Glu OAAl + Asp PROT + Asp PROT + ACO PROT ".* SUCl PROT "* FUMl PROT + Ala PROT "* Glu

0.023 0.068 0.42 0.002 0.28 0.53 0.14 -0.001 0.010 -0.005 -0.005 0 0 -0.012 -0.046 0.020 -0.013 -0.004 0.020 0.009 0.036 0.21 0.037 0.016 0.13 0.044

0.017 0.047 0.56 0.003 0.23 0.48 0.11 0 0.004 0 0.005 0 0 0.015 -0.031 0.015 -0.006 0 -0.008 0.003 0.051 0.11 0.054 0.022 0.11 0.051

0.034 0.054 0.18 0.001 -0.37 -0.52 0.14 -0.002 0.006 -0.014 -0.015 0 0 -0.023 -0.058 0.011 -0.019 -0.013 0.012 0.005 0.025 0.12 0.012 0.018 0.078 0.028

0.026 0.036 0.24 0.002 -0.32 -0.42 0.11 -0.002 0 -0.007 -0.006 0 0 0.009 -0.040 0.008 -0.012 -0.006 -0.017 -0.001 0.048 0.029 0.036 0.026 0.064 0.039

1.91 Sum -0.31 -0.16 1.84 "Enzyme activitychanged to (0.80, 0.85, 0.90, 0.93, or 0.95) X original steady-state value. 'Enzyme activity changed to (1.05, 1.07, 1.10, 1.15, 1.20, or 1.30) X original steady-state value. Stimulus: enzyme changed in activity; numbers refer to reactions in Fig. 2. C;lcyc'eC02: flux control coefficient as defined in Table 5. e C?ta1C02: flux control coefficient as defined in Table 5.

by Establishment of a New Steady State-The processes which a metabolic system is controlled and steady state is reestablished after perturbations are of considerable interest. Therefore, the time-dependent changes metabolite in concentrations and fluxes were analyzed after smallincreases in the activity of either malic enzyme or malate dehydrogenase. It was noted that many fluxes and metabolite concentrations had reached steady state by 2 min (Fig. 3). Except for their common metabolite,malate,these two enzymes exhibited nearly equal and opposite affects on the system's responses. This is reflected by the nearly equal andopposite cycle flux coefficients, C%z (for malate dehydrogenase, - 0.18 and for malicenzyme, 0.24). However, whenlarger changes were made, C@z had the same sign. Direct examination of the coefficients and the structureof the model did not reveal the mechanism of this sign change. However, by examining the changes in individual fluxes and metabolite concentrations over time,themechanism could beunderstood. The sign change occurred because of the change in affect of pyruvate concentration on reaction 16 (Miniprint). Since directmeasurement of control coefficients cannot reveal how system variables are affected by perturbations over timeor how steady state is reestablished,metabolic models provide an additionaladvantage over in vivo measurements,as small time frames canbe examined in detail. A realistic model of the tricarboxylic acid cycle has allowed us to test implications of MCT. This model does generate the expected summation properties for flux and for most metabolite control coefficients. However, changes of 2% or less above and below the operating point were necessary for the reattainment of steady state andfor the accurate calculation of control coefficients. This analysis provided a systematic way to predict rate-controlling points for models simulating changes influx, metabolite, and enzyme concentrations which occur during environmental stress or during metabolic transformations such as differentiation or aging. Because these highly data-based metabolic models have demonstrated predictive value for in vivo metabolism (see companion paper), control analysis of these models should provide an accurate assessment of the distribution and mechanism of control for the in vivo pathway.
REFERENCES 1. Higgins, J. (1963) Ann. N. Y. Acad. Sci. 108, 305-321 2. Kaicser, H. and Burns, J. A. (1973) in Rate Control of Biological Processes (Davies, D.D., ed)Symposium of the Society for Experimental Biology 27, pp. 65-104, CambridgeUniversity Press, London 3. Heinrich, R., and Rapoport, T. A. (1974) Eur. J. Biochem. 42, 89-95 4. Heinrich, R., and Rapoport, T. A. (1974) Eur. J. Biochen. 42, 97-105 5. Rapoport, T. A., Heinrich, R., Jacobasch, G., and Rapoport, S. (1974) Eur. J. Biochem. 42, 107-120 6. Crabtree, B., and Newsholme, E. A. (1985) Curr. Top. Cell. Regul. 25,21-76 7. Savageau, M. A. (1976) Biochemical Systems Analysis: A Study of FunctionandDesign in Molecular Biology, Addison-Wesley, London 8. Sorribas, A., and Savageau, M. A. (1989) Math. Biosci. 94, 195238 9. Torres, N. V., Mateo, F., Melendez-Hevia,E., and Kacser, H. (1986) Biochem. J. 234, 169-174 10. Torres, N. V., Mateo, F., and Melendez-Hevia, E. (1988) FEES Lett. 283,83-86 11. Cornish, A., Greenwood, J. A., and Jones, C. N. (1988) J. Gen. Microbiol. 134, 3111-3121 12. Brindle, K. (1988) Biochemistry 27,6187-6196 13. Nimmo, H. G., and Cohan, P. T. W. (1987) Biochem. J. 247, 113

than having to calculate unfamiliar elasticities, MCT would be more widely used experimentally. Atkinson (47) attempted such an analysis, assuming mass action enzyme kinetics. This type of analysis was used for the tricarboxylic acid model, relating Vv,v, and flux. However, it did not predict the measured control coefficients. In the present study, an analysis similar to thatof Atkinson was done, assuming that enzyme kinetics followed the Michaelis-Menten equation. This kinetic equation is frequently used as a first step in the experimental analysis of an enzyme to determine an apparentK,. From this analysis the following correlations were found 1) if S/K, 2 1, then 2 5 V / J I1 and 0.5 5 Cy 5 1.0; 2) if S/ K , < 1, then V/J > 2 and CUJ< 0.5. Although all of the enzymes modeled are described by more complexmechanisms, these ratios were calculated for all enzymes to see if they For some enzymesthere was good predicted themeasured CUJ. correlation between enzymeparameters and Cy,but for others there was not (Table8). Because this method was not predictive, a further analysis was applied in which the complex mechanisms used in the model were "reduced" to a MichaelisMenten equation (Table 8).An analysis of the enzymes based on relaxation constants as suggested by Heinrich and Rapoport (3) was also performed. None of these methods consistently predicted the size of the flux control coefficient (see Miniprint).

3112

Control Analysis of Tricarboxylic Acid Cycle in D. discoideum

30. Cascante, M., Franco, R., and Canela, E.I. (1989)Math. Biosci. 94,271-288 31. Cascante, M., Franco, R., and Canela, E.I. (1989)Math. Biosci. 94,289-310 32. Sauro, H. M., Small, J. R., and Fell, D. A. (1982)Eur. J.Biochem. 166,215-221 33. Hofmevr. J. H. S.. Kacser. H.. and van der Merwe. K. J. (1986) . . Eur. "J.'Biochem. 156,631-641 34. Kacser, H., and Burns, J. A. (1981)Genetics 97,639-666 35. Keightley, P. D., and Kacser, H. (1987)Genetics 117,319-329 36. Sherwood, P., Kelly, P., Kelleher, J. K., and Wright, B. E.(1979) Comp. Prog. Biomed. 10,66-74 37. Wright, B. E., and Kelly, P. J. (1981)Curr. Top. Cell. Regul. 19, 103-158 38. Wright, B. E. (1984)J. Theor. Biol. 110,445-460 and Reimers, J. M. (1988)J. Biol. Chern. 163, 39. Wright, B. E., 14906-14912 40. Park, D. J. M., and Wright, B. E. (1973)Comp. Prog. Biomed.3, 10-26 41. Wright, B. E., Tai, A., and Killick, K. A. (1977) Eur. J. Biochem. 74,217-225 42. Wright, B. E., and Butler, M. H. (1987)in Evolution and hngeuity in Animals (Woodhead, A. D., and Tomson, K. H., eds) pp. 111-122,Plenum Publishing Corp., New York 43. Kelly, P. J., Kelleher, J. K., and Wright, B. E . (1979)Biochem. J. 184, 581-588 44. Kelly, P. J., Kelleher, J. K., and Wright, B. E. (1979)Biochem. J. 184,589-597 45. Canela, E. I., Ginesla, I., and Franco, R. (1987)Arch. Biochem. Biophys. 264,142-155 46. Rigoulet, M., Averet, N., Mazed, J. P., Guerin, B., and CoLadon, F. (1988)Biochim. Biophys. Acta 932,116-123 in Control of Metabolic Processes (Cornish47. Atkinson, D. E.(1990) Bowden, A., and Cardenas, M. L., eds) NATO AS1 Series, pp. 3-11, Plenum Publishing Corp., New York

14. Walsh, K., and Koshland, D. E.,Jr. (1985)Proc. Natl. Acad. Sci. U.S .A. 82,3577-3581 15. Albe, K. R., Butler, M. H., and Wright, B. E. (1989)J. Theor. Biol. 143,163-195 16. Keleti, T., and Ovadi, J. (1988)Curr. Top. Cell. Regul. 29, 1-33 17. Clarke, F. M., and Masters, C. J. (1975)Biochim. Bioplzys. Acta 381,37-46 18. Welch, G. R. (ed) (1985)Organized Multienzyme Systems Catalytic Properties, Academic Press, Orlando, FL 19. Wright, B. E.,and Albe, K. R. (1990) in Control of Metabolic Processes (Cornish-Bowden, A., and Cardenas, M. L., eds) NATO AS1 Series, pp. 317-328,Plenum Publishing Corp., New York 20. Groen, A. D., Wanders, R. J. A., Westerhoff, H. V., Van der Meer, R., and Tager, J. M. (1982)J. Biol. Chem. 257, 27542757 21. Kholondenko, B., Zilinskiene, V., Borutaik, V., Ivanoviene, L., Toleikis, A., and Praskevicius, A. (1987)FEBS Lett. 223,247250 22. Rigoulet, M., Guerin, B., and Denis, M. (1987)Eur. J. Biochem. 168,275-279 23. Brand, M. D., Hafner, R. P., and Brown, G. C. (1988)Biochem. J. 265,535-539 24. Petronilli, V., Azzone, G. F., and Pietrobon, D. (1988)Biochim. Biophys. Acta 932,306-324 25. Middleton, R. J., and Kacser, H. (1983)Genetics 105,633-650 Dean, A. M., and Hartl, D. L. (1987)Genetics 26. Dykhuizen, D. E., 115,25-31 27. Kacser, H., and Porteous, J. W. (1987)Trends Biol. Sci. 12, 514 28. Barrett, J. (1988)Parasitology 97,355-362 29. Hofmeyr, J. H. S. (1986)C a b s 2,5-11

SUPPLEMENTAL HATERIAL

OItTYOSTELIUn QlXQlRM: 11.

TO SYSTEMS ANALYSIS OF THE TRICARBOXYLIC ACID CYCLE

CONTROL ANALYSIS
TABLE I: PCRCtll,'

IN

Kathy R. Albe and Barbara E. Yrlght

"LRIITIOH I N "LTLIBOLITI CONCINTWIIBN BETYttN T.9 Lho 1-11 "IWUTtl

values generated after a ten minute rlmulation.

FIGURE 1: CALCULATION OF CONTROL COEFFICIENTS
0

I n stimulus. The slope of ihe line. fltted byregrelslon, i s the control caefflcient. line~

Control Analysis of TricarboxylicAcid Cycle in D. discoideum

3113

Since t h e P a t i o s determined from the basic Michaelis-Menten equation were unambiguous and apparently predictive of C'., the more complex equations Used in t h e nvldel were reduced to t h i s form. To develop these reduced equations, a l lm e t a b o l i t e s except S,md f l u x were considare4 t o be constant, and new valuer f e r Km and V #ere c a l c u l a t e d . T h i s a n a l y s i s was a p p l i e d to a11 the enzymes r n Table 8 and t h e new ratlor, and V"/Ji, were analyzed a s p r e d i c t ~ r ~o f cp and cF7'". As w i t hA t k i n s o n ' s approach, n e i t h e ro ft h e s ea p p l o a c h e Ip r e d i c t e dt h e size af the control coefficient<.

TABLE 8: FACTORS AFFECTING OBSERVED

FLUX CONTROL COEFFICIENTS IC',) S/Km

V/J,

STIMULUS'
1 GlUOH

d ' '
1.0

cfe''*'
0.027

SUBSTRATE GLU 2KG1

SUCI

S/Km'"
1.1

V"/J; 1.7 16.9 1.3~10'

3.0 8.0
0.10

6.5

2 2-KGOC
3
5 6
SOH
1 Fumarase

0.12
0.66

0.063
0.35

750.01 0.01 3157

1.1
25.7

0 0.24
0.72

0 .O.l8 0.2k 0
-0.011

FUMl

8.0 0.33
0.77 0.59

1.1
9.0

0.029
3.5~10" 2.9~10"

MOH

ME

MALI MAL1
ASP PYR ACO IC0 OM2 ISOC ASP

0.16 0.59

44.2 2.8
168 1.1 4.1

1.6
2.7

0.13
5.WV 2.3~10'

Is'
2.1
6.0
1.7 0.05'

1.1
1.7

8 P O C II
I3
15 16

0
0.025

20
1.1

cs
IsocOH
AspTA AlaTA

11.3

0.36
0.077 4.0

0 0.69
0.12

0
-0.054

136
11.1 41.4

0.48 0.043

3.1
23.7

4.8~10"

8.4
0.010
4

ZKGI
0.011
PYR

0.025
0.34

1.2 30
1.7

8.3~10.'
0.011

5.3 5.3

I:
2:

STIMULUS: Enzyme

GLU 0.40 changed i n activity; numberr r e f e r

11.2 to reactions i n Figure 2.

0.10

c?

: F l u xc o n t r o lc o e f f i c i e n tf o rm s p o n l e of J , . to stimulus, i,where J , i s the f l u ~ e s p e n s e a f t h e r e a c t i o n c a t a l y z e d by enzyme 1. and i i s t h e e n q m changed I n activity.

3:

c~cyc'' :
S/K.":

Flux controc l oefficlent for response Jcyclc. to r t l m u l u r , 1, where Jcycle i s t h e sum o f t h e f l u x responses o f reactions 2 and 13. and i i o the enzym changed in activity. The ratio o fs u b s t r a t e
concentration

4:

to the K." fromthereducedequation

equation to f l u x .J

I: V/J,:
6: 7:

The ratio o f enzyme activity, V",

from the reduced

s : Relaxation constant

a$ d e f i n e d by H e i n n c h 1 Rapoport 13)
a reducedequationderived
1

Ratiosdeternlnsdfrom 19partate. Ratio3 determined from

for t h e a l l o r t e r i c e f f e c t o r ,

8:

r e d u c e de q u a t i o nd e r i v e di n

terms of I / a c e t y l COA

IN THE RE-ESTABLISHMENT OF STEADY-STATE: Examination of the TIME EVENTS AND MECHANISMS control c o e f f i c i e n t s are generated i s of p a r t i ~ ~ l a interest. r m e c h a n l mb yw h i c ht h e s ef l u x The m o s t intriguing case is at t h eb r a n c h p o i n tm e t a b o l i t e ,m a l a t e .E x a m i n a t i o no ft h ec i t r a t e synthase reaction mechanism, r e v e a l s t h a t o . x a l o l c e t a t e i s mom rate l i m i t i n g (S/h 0.36) than observed r a t e O f t h i s reaction. but increasing t h e rate s f a c e t y l CoA (S/h I 6 . 0 ) to the oxalascetate synthesis through malate OH r e s u l t e dI n a negatqvecycle flux c o e f f i c i e n t . The c y c l ef l u xc o e f f i c i e n t i o t h e sum ofthe CO, generated a t r e s c t i o n l 2 and 13. Therefore, the decrease in CO, fluxfrom reaction 2 m u s t be greater thanthe ~ncreasefrom r e a c t i o n 13. This

I S confirmed by the

f l u xc o n t r o lC o e f f i c i e n t sf o r

each

reaction,

C g - -0.45

C C ~ " " ' -

C C ~ ' x 2 . 0 * C~*x2.4121/4.414

CE's

0.15

and

-0.18; Table 5 ) .

Increasing

the rate o f s y n t h e s i s o f a c e t y l CoA t h r o u g h i n c r e a s i n g t h e e n z y m a t i c a c t i v i t y o f m a l i c enzyme. caused the opposite a f f e c t on c y c l ef l u x , 3 . e. an increase i n t h e generation o f LO,

c " i :

= -0.Zl;C~'

0.52).

The mechanisms f o r there results

are not obvlour from the

3114
FIGURE 3:

Control Analysisof Tricarboxylic AcidCycle in D. discoideum

PERCENT CHANGE I N CONCENTRATION OR FLUX IN RESPONSE TO INCREUSED MALATE DEHYDROGENASE OR I U L I C ENZYME UCTIVITY'

3c

30

FIGURE 3C. Response t o MOH: PYR ( e ) : K O (0);RX16 (i). Response to H i PYR (AI; ACO (A); RX16 (GI.

FIGURE 30: Response to HOH: 2KGI (t): R X Z ( 0 ) ; RXE (A). R e r p o n r e to ME: ZKGI ( 4 : RX2 (0);RXE ( 0 ) .