THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistryand Molecular Biology, Inc.

Vol. 267, No. 5,Issue of February 15,pp. 3101-3105,1992 Printed in U.S.A.

Systems Analysis of the Tricarboxylic Acid Cycle in Dictyostelium discoideum

I. THE BASIS FOR MODEL CONSTRUCTION*
(Received for publication, November 5, 1990)

Barbara E. Wright, Margaret H. Butler, and KathyR. Albe

From the Division of Biological Sciences, University of Montana, Missoulu, Montana 59812

artifacts (19-22) and to be incompatible with the other parameters in the enzyme kinetic expressions, namely, the rates of the reactions, metabolite concentrations, and kinetic constants. The values to replace Vmax were therefore calculated for each equation as the only unknown, assuming the other parameters to be correct. These calculated values are called Vvivo. Reaction rates in vivo were determined in a number of ways and were very reliable. The ratesof individual reactions were based on the rateof accumulation of counts in a product knowing the specific radioactivity of the immediate precursor (5, 23, 24). These values were substantiated using a specific radioactivity curve-matching program (TFLUX (25)) to simulate data in which the specific radioactivity of many intermediates in the network was followed as a function of time after exposure to tracer (13, 14) or perturbing (2) levels of [4C]glucose. Asthese analysesinvolved many interdependent metabolites and reactions, they also provided information An intriguing question for biochemists is the extent to regarding compartmented pools. For example, a difference in which enzyme activities, metabolite concentrations, kinetic mechanisms and constantsdetermined i n vitro apply in vivo. the labeling patterns of UDP-glucose and glucose 1-phosphate One approach to this problem is to construct metabolic models required the existence of two pools of glucose 1-phosphate (2) already been established that UDP-glucose was a single that incorporate such in vitro data and then to determine how (it had well the behavior of the model describes metabolism in the pool (14)). Other studies directly demonstrated the location living organism. This in turn can only be judged by the of the two pools of hexose phosphates (13). Knowing total predictive value of the model. Making a model match a given substrate concentrations, these analyses gave the concentradata set does not necessarily indicate that it reflects reality; tions of the metabolite compartments. As most metabolites the model must be sufficiently realistic to have predictive are relatively stable, their total concentration can be detervalue. When predictive value has been demonstrated, it is mined quite accurately. In comparing metabolite and enzyme reasonable to conclude that the model parameters may ap- concentrations in seven different organisms, metabolite concentrations were found to be more consistent, as might be proximate these operative in vivo. values are also relatively consistent In 1967 a simple metabolic model was constructed simulat- expected (26). K,,, and Ki ing the glycogen cycle and saccharide end-product synthesis and areoften similar for a given enzyme isolated from differin Dictyostelium discoideum. Five assumptions (predictions) ent organisms. For example, for the pyruvate dehydrogenase in thismodel were later demonstrated to be correct (1). Over complex in Dictyostelium (27), Ascaris (28), and cauliflower the years, about 40 predictions based on increasingly complex (29), respectively, the K,,, values (mM) for pyruvate were 0.14, models of metabolism in thissystem have been substantiated, 0.18, and 0.20; for CoA,0.008,0.005, and 0.007; for NAD, three unknowingly in otherlaboratories (1-17). The program 0.11, 0.01, and 0.12; the Ki values (mM) for NADH were 0.049, used in the construction of these realistic models is called 0.025, and 0.034; for CoA, 0.024, 0.062, and 0.013. For malate METASIM (18),which contains 27 different enzyme mecha- dehydrogenase in Dictyostelium (30), pig heart (31), mitonisms; the user indicates the appropriate onefor each reaction chondrial bovine heart (32), soluble bovine heart (32), and and supplies the kinetic constants. Other input data are the Bacillus sdtilis (32), respectively, the K , values (mM) for flux relationships and compartmented metabolite concentra- malate were 1.33,0.8,0.99,0.54, and 0.90; for NAD, 0.10,0.20, tions in the metabolic network. Most of the enzymes depicted 0.54, 0.20, and 0.4; for oxaloacetate 0.27, 0.04, 0.04, 0.05, and in the models were isolated from Dictyostelium, purified and 0.06; for NADH, 0.04, 0.02, 0.02, 0.04, and 0.03. The relative characterized using standard in vitro methods. consistency of metabolite concentrations and kinetic conIn these models with demonstrated predictive value, Vmax stants among different organisms suggested that these values values could not be used, as theywere frequently found to be were reliable as input datafor metabolic models. In contrast toreaction rates, kinetic constants, metabolite * This work wassupported by National Institutes of Health Public concentrations and compartments, there are a number of Health Service Grant AG03884. The costs of publication of this article values to enzyme were defrayed in part by the payment of page charges. This article reasons to question the relevance of Vmax must therefore be hereby marked advertisement in accordance with activity in intact cells. Cellular organization is destroyed in the preparation of extracts, membrane-bound enzyme com18 U.S.C. Section 1734 solely to indicate this fact.

A steady-state model of the tricarboxylic acid cycle w a s constructed using a dynamic systems analysis computer program, METASIM. The model was based on radioactive tracer analyses which provided flux relationshipsandcompartmentedmetaboliteconcentrations. Ten of the enzymes modeled were purified and Dictyostelium discoideum. Alcharacterized from though experimentally determined enzyme mechanisms and constantswere used in the model, Vmax values were found to be unreliable, i.e. they did not reflect enzyme activity in vivo.This value was therefore calculated as the only unknown in each enzyme kinetic equation and called Vvivo,to distinguish it from Vmax determined in vitro.

3101

3102

Model of Tricarboxylic Acid Cycle in Dictyostelium

FIG. 1. A steady-state model of the citric acidcyclein D. discoideum. Encircled reactions are those for which the enzyme from D. discoideum has been kinetically characterized. All metabolite pools are intramitochondrial, as defined by Kelly et al. (37). See Experimental Procedures for further details. Glu, glutamate; 2KG, 2-ketoglutarate; SUC, succinate; FUM, fumarate; MAL, malate; OAA, oxaloacetate; CZT, citrate; ASP, aspartate; ALA, alanine; PROT, protein; PYR, pyruvate; ACO, acetyl-coA.

TABLE I1 plexes are disrupted; enzymesites bouna by product or inhibA comparison of calculated enzyme activity in vivo, Vuiuo, and V,, itor in vivo (33, 34) may become freed and available to values for seven enzvmes of the tricarbonvlic acid cvcle substrate on dilution in extracts; unknown inhibitors or acEnzvme V. V; Ref. tivators may be diluted,destroyed, or created; proteolytic inactivation may be initiated by extract preparation (19, 20); mM/min mM/min andfinally,enzymeactivity is frequentlymeasuredunder 1.8 40 Isocitrate DHb 271.0 optimal rather than physiological conditions of pH, temper2-Ketoglutarate DC 4.8 41 7,608.0 196.0 Malate DH 77.8 30 ature, cofactor and protein concentration. As proteins, enMalic enzyme 8.6 39 3.08 zymesmayplayrolesother thanbeing catalysts. Proteins 45.0 Succinate DH 3.15 42 may be used as an energy source, especially under nutritional Citrate synthase 8.23 43 1,071.0 stress. Because of the general vulnerability of most proteins 2.1 27 Pvruvate DC 258.0 to proteolytic attack,excessive enzyme protein concentration Converted to m M mitochondrial volume, using the conversion may beessential to ensure adequate catalytic activity in times relationships: 1 mg of protein = 1.4 mg of dry weight; pmol/min/mg of stress. Catalytically active enzymes have also been found dry weight X 150 = m M packed cell volumein amoebae (44), and cell/ to serve structural roles (35). Regardless ofthe specific events mitochondrial volume is 1/5. DH, dehydrogenase. occurring in uiuo, treating VmaX as the unknown in each e DC, dehydrogenase complex. enzyme kinetic expression and calculating Vvivoresultedin models with predictive value. This approachwastherefore used to construct the model of the tricarboxylic acid cycle. to operate and maintain steady-state levels of cycle intermediates,
~ ~

EXPERIMENTAL PROCEDURES

In this model (Fig. l ) , flux through the cycle was consistent with

O2consumption, net protein degradation (the energy source in this

system), and NH, production (36, 37). Flux into the cycle (reactions 21-26, Fig. 1) was based on an amino acid analysis of Dictyosteliurn protein at three stages of differentiation (3). The percent concentration of each amino acid in average protein was the same at each stage, indicating an even use of available protein, i.e. comparable oxidation rates of individual amino acids over the course of development. Knowing the pathways by which each amino acid is converted to one or more cycle intermediates (3, 38), the flux of each amino acid into specific intermediates could be calculated (37) and must sum to the flux through the cycle (reactions 21-26, Fig. 1). In a system using only amino acids as the source of cycle intermediates, more amino acids are converted to four- and five-carbon intermediates than to acetyl-coA. For the citrate synthase reaction Tables 1, 3, 4, 5, and 6 are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of astandard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

the flux of acetyl-coA into the cycle must equal that of oxaloacetate. Therefore a pathway is required which converts excess four- or five-carbon intermediates to acetyl-coA. The pathway for converting tricarboxylic acid cycle intermediates to acetyl-coA involves malic enzyme. In this reaction, malate is decarboxylated to yield pyruvate which is in turn decarboxylated to form acetyl-coA via the pyruvate dehydrogenase complex. This pathway has been shown to occur in Dictyostelium, and both of these enzymes have been purified and kinetically characterized (27, 39). We have purified and characterized seven of the nine enzymes essential to the cycle (Fig. 1 and Table I): 2-ketoglutarate dehydrogenase complex, succinate dehydrogenase, malate dehydrogenase, malic enzyme, pyruvate dehydrogenase complex, citrate synthase, and isocitrate dehydrogenase. These enzymes are listed in Table 2, together with their V,,, values and their calculated V,i, values. Two of the most striking discrepancies between the V,i, and VmaX values are for the enzyme complexes, 2-ketoglutarate dehydrogenase complex and pyruvate dehydrogenase complex. As these complexes are membrane bound, they dissociate during extract preparation, and higher Vvivo compared with VmaX values might have been anticipated. Most isolated preparations of succinate dehydrogenase containa deactivated form of the enzyme bound tightly to oxaloacetate (34), preventing succinate from binding. As purification procedures can

Model of Tricarboxylic Acid Cycle in Dictyostelium

activate the inactive form,a V , . value 10-fold higher than the V,,, value is not surprising. The V,, and Vvivo values for malate dehydrogenase and malic enzyme are reasonably close, but there no is obvious for isocitrate dehydroexplanation for the differences in these values genase or citrate synthase. It is of coursepossible that unknown activators or inhibitors are present in uiuo. Calculated Vvivovalues could include correction factors for kinetic constants as well as for V , . However, evidence exists that kinetic constants used in the carbohydrate models approximate those operative in vivo (see Dis-

3103

cussion).

RESULTS

The METASIM modelshown in Fig. 1 was constructed based on an original TFLUX analysis of the tricarboxylic acid cycle in D. discoideum (36, 37). This model is highly constrained since many related intermediates were isolated and their interdependent specific radioactivities determined. All single poolsincluded in the original TFLUX modelwere assumed to exist entirely within the mitochondrion. For those metabolites that existed in two or more pools,such as malate, the pool external to the cycle was considered to be cytosolic and was eliminated from the present analyses. In these cases, the mitochondrial pool concentration predicted by the TFLUX model was used rather thantotal metabolite concentration. Theenzyme mechanisms and kinetic constants used in the model are given in Tables 3 and 4. To consider intramitochondrial concentrations and fluxes, mitochondrial volume wasassumed to be 20%of the totalcell volume (46). Cycle flux, determined from net protein degradation, or oxygen consumption (36, 37) was approximately 0.4 mM/min based on total cell volume. Therefore, based on mitochondrial volume, cycle flux is 2 mM/min. Amino acids serve asan energy source inthis organism (3), andthe constant influx of carbon is balanced by the efflux of carbon through the generation of COz. A number of structural constraints exist on the fluxes in a metabolic pathway in steady state when metabolite concentrations and fluxes are constant over time (3, 37). For each metabolite, the requirement that the input flux equals the output flux imposescertain constraints on the relative values of the fluxes (namely that the vector of steady-state fluxes must lie in the null space of the stoichiometric matrix of the metabolic system). In this case, there are 23 net fluxes (described by26 reactions) and 13 variable metabolites; rank analysis of the stoichiometry matrix confirms that there are no moiety conservation relationships among the metabolites, so the dimension of the null space is 10 (23 fluxes - 13 metabolites); that is, there are only 10 independent fluxes, and the remaining 13 can be determined from these. Six of the independent fluxes are the input reactions from protein degradation and were determined directly from experimental measurements (3). That the reaction pairs 9 and 10, 14 and 19, and 18 and 20 have zero net flux at the observed steady state accounts for a further three degrees of freedom. Thus, of the total 26 reactions, 13 can be specified. The 13 independent fluxes wereconsidered to be 9,10,14,16,18-26 (Fig. 1).Table 5 shows the net reaction rates used in the steadystate tricarboxylic acid model compared with similar rates from the TFLUXanalysis. The metabolite concentrations are shown in Table 6 and were the same as thevalues determined from the TFLUX analysis adjusted for mitochondrial volume (36, 37). Table 1gives the enzyme mechanisms and kinetic constants determined in vitro for enzymes purified from D. discoideum. These data were used in the model (Table 3), supplemented (as required by the mechanisms) bysome assumed values

whichcould not be obtained experimentally. A dead-end competitive inhibition mechanism (Table 4) rather than a rapid equilibrium-ordered BiBi mechanism (Table 1) was used for three enzymes (glutamate dehydrogenase, isocitrate dehydrogenase, and citrate synthase) to include product inhibitions which were physiologically significant (40, 43, 48). As discussed earlier, experimentally determined Vmax values were not used, but rather a calculated value called Vvivo (Vl, V2 in Table 3) was used for maximal enzymatic activity in these models. Flux and metabolite concentrations over the time course of the simulation are shown in Table 6. The concentrations of NAD, NADH, and CoAwere fixed time constants, taken from experimental measurements (45), and the fixed input rates (reactions 21-26)were based on the constant rate of net protein degradation and calculated rates of conversion of individual amino acids to specific cycle intermediates (3,37). Over a 10-min simulation period the flux and metabolite concentrations vary less than 0.04%, indicating steady-state conditions. The definition of steady state is discussed in the companion paper.
DISCUSSION

* D. Fell, personal communication.

In the model of the tricarboxylic acid cycle, flux was based on 0 consumption, a TFLUXanalysis, the net rateof protein degradation, and the rate of conversion of amino acids to specific cycle intermediates. In terms of mitochondrial volume the rate of conversion of malate to pyruvate (reaction 6) was 1.09 mM/min, and therate of pyruvate formation from amino acids (reaction 25) was 0.45 mM/min, giving a combined rate of 1.54 mM/min for reaction 8, which is catalyzed by pyruvate dehydrogenase. This rate was also checked independently in vivo. The pyruvate dehydrogenase reaction in the slime mold, assayed in vitro using [l-4C]pyruvate and quantitating the evolution of 14C02, can be examined with virtually no interference from other enzymatic reactions (49). Pyruvate carboxylase, known to interfere with the pyruvate dehydrogenase assay (38), has not been detected in D.discoideum, and glycolysis has been shown to play only a minor role. Significantly, the pyruvate dehydrogenase reaction assayed with [l-14C]pyruvatein crude extracts was completely dependent upon NAD, coenzyme A, and thiamine pyrophosphate, suggesting that pyruvate was converted to 14C02only via the reaction catalyzed by pyruvate dehydrogenase (27). In all likelihood this is also true of the reaction measured in vivo. The in uivo rate of the reaction catalyzed by pyruvate dehydrogenase was measured by exposing cells to [l-C]alanine and relating the rate of 14C0, evolution to the specific radioactivity of the isolated [l-4C]pyruvate (49). The rate obtained, 1.65 mM/min, is in excellent agreement with the rate obtained using the othertypes of analyses summarized above. Inthe METASIM models of carbohydrate metabolism there is evidence that many of the K,,, values used do reflect K , values operative in vivo. Perturbation studies have been carried out in which Dictyostelium was exposedto glucose, Pi, uracil, or uridine, for example, and compared with the models perturbed by these same metabolites (1, 10, 11).These perturbations resulted in changes in the levels of other metabolites, such as glucose 6-phosphate, UDP-glucose, glycogen and trehalose, in boththe models and Dictyostelium. The similarity of these effects indicate similar substrate-K,,,relationships in themodels and the organism. For example, glucose perturbation results in approximate 3-fold increases in glucose 6phosphate and trehalose levels and less than a 2-fold increase in UDP-glucose levels (1, 11).The increased trehalose level results from the relationships between the K , values of tre-

3104

Model of Tricarboxylic Acid Cycle Dictyostelium in

20. Wright, B. E., and Dahlberg, D. (1968) J. Bucteriol. 95,983-985 21. Gustafson, G.L., and Wright, 3- E. (1972) Crit Rev. Microbial. 1,453-478 22. Gezelius, K., and Wright, B. E. (1965) J. Gen. Microbiol. 38,309327 23. Pannbacker, R. G. (1967) Biochemistry 6,1287-1293 24. Marshall, R., Sargent, D., and Wright, B. E. (1970) Biochemistry 9,3087-3094 25. Sherwood, p., Kelly, p. J.9 Kelleher, J. K.9 and Wright, B. E. (1979) Comp. Prog. Biomed. 1 0 , 66-74 26. Albe, K. R., Butler, M. H., and Wright, B. E. (1989) J. Theor. Biol. 143, 163-195 27. Butler, M. H., Mell, G. P., and Wright, B. E. (1985) Curr. Top. Cell. Regul. 26, 337-346 28. Komuniecki, R., Komuniecki, P. R., and Saz, H. J. (1979) Biochim. Biophys. Acta5 7 1 , 1-11 29. Randall, D.D., Rubin, P. M., and Fenko, M. (1977) Biochim. Biophys. Acta 4 8 6 , 336-349 30. Emyanitoff, R. G., and Kelly, P. J. (1982) J. Gen. Microbiol. 1 2 8 , 1767-177i 31. Raval, D. N., and Wolfe, R. G. (1962) Biochemistry 1,263-269 32. Thomas E. Barman (ed) (1969) Enzyme Handbook SpringerVerlag, New York 33. Bloch, W., MacQuarrie, R. A., and Bernhard, S. A. (1971) J. Biol. Chem. 246,780-790 34. Ackrell, B. A. C., Kearny, E. B., and Singer, T. P. (1978) Methods Enzymol. 5 3 , 466-483 35. Wistow, G. J., Mulders, J. W. M., and de Jong, W. W. (1987) Nature 326,622-624 36. Kelly, P. J., Kelleher, J. K., and Wright, B. E. (1979) Biochem. J. 184,581-588 37. Kelly, P. J., Kelleher, J. K., and Wright, B. E. (1979) Biochem. J. 184,589-597 38. Palmer, T. N., and Sugden, M. C. (1983) Trends Biochem. Sci. 8 , 161-162 39. Kelleher, J. K., Kelly, P. J., and Wright, B. E. (1979) J. Bucteriol. 138,467 40. Emyanitoff, R. G. (1982) Exp. Mycol. 6 , 274-282 41. Heckert, L. L., Butler, M. H., Reimers, J. M., Albe, K. R., and Wright, B. E. (1989) J. Gen. Microbiol. 135, 155-161 42. Butler, M. H. (1989) Exp. Mycol. 1 3 , 294-298 43. Porter, J. S., and Wright, B. E. (1977) Arch. Biochem. Biophys. 181,155-163 44. Walsh, J. W., and Wright, B. E. (1978) J. Gen. Microbiol. 108, 57-62 45. Owen, T. G., and Hochachka, P. W. (1974) Biochem. J. 1 4 3 , 541-553 46. Srere, P. A. (1967) Science 158,936-937 47. Wright, B. E., and Wasserman, M. E. (1964) Biochim. Biophys. Acta 90,423-424 48. Komuniecki, P. R., Detoma, F. J., and Wright, B. E. (1979) Abstracts of the Annual Meeting of the American Society of Microbiology, p. 103 49. Butler, H.M., and Wright, B. E. (1989) Biochim. Biophys. Acta 991,337-339 50. Landridge, W. H. R., Komuniecki, P., and DeToma, F. J. (1977) Arch. Biochem. Biophys. 178,581-587

halose-6-phosphate synthase for glucose 6-phosphate and UDP-glucose and the concentrations of these substrates in the cell and in themodel. Perturbation studies will be carried out with the tricarboxYlic acid cyclemodel and the organism to test the many assumptions and predictions inherent in the model. Constraints on the model will also be examined, to determine if model behavior is affected by changes in enzyme mechanisms, configuration, kinetic constants, andso on.
Acknowledgments-We would like to thank Dr. David Fell for his considerations of our problems in meeting the requirements for the steady state in these models and kindly analyzing our pathway for us. We would also extend thanks to the organizers of the symposium on metabolic control held in El Ciocco, Italy, in 1989 for providing us with a forum for presenting these ideas and interacting with other researchers in the field. REFERENCES 1. Wright, B. E., and Kelly, P. J. (1981) Curr. Top. Cell. Regul. 1 9 , 103-158 2. Wright, B. E., and Reimers, J. M. (1988) J. Biol. Chem. 2 6 3 , 14906-14912 3. Wright, B. E., and Butler, M. H. (1987) in Evolution and Longeuity in Animals (Woodhead, A. D., and Thomson, K. H., eds) pp. 111-122, Plenum Publishing Corp., New York 4. Wright, B. E., and Park, D. J. M. (1975) J. Biol. Chem. 2 5 0 , 2219-2226 5. Sargent, D., and Wright, B. E. (1971) J. Biol. Chem. 246,53405344 6. Wilson, J. B., and Rutherford, C. L. (1978) J. Cell Physiol. 9 4 , 37-46 7. Wright, B. E. (1968) J. Cell Physiol. 7 2 , (Suppl. l ) , 145-160 8. Wright, B.E., and Dahlberg, D. (1967) Biochemistry 6 , 20742079 9. Wright, B. E., and Marshall, R. (1971) J. Biol. Chem. 246,53355339 10. Wright, B. E., Tai, A., and Killick, K. A. (1977) Eur. J. Biochem. 74,217-225 11. Wright, B. E., Tai, A., Killick, K. A., and Thomas, D. A. (1979) Arch. Biochem. Biophys. 192,489-499 12. Wright, B. E. (1984) J. Theor. Biol. 110, 445-460 13. Chiew, Y. Y., Reimers, J. M., and Wright, B. E. (1985) J. Biol. Chem. 260,15352-15331 14. Wright, B. E., Thomas, D. A., and Ingalls, D. A. (1982) J. Biol. Chem. 267,7587-7594 15. Emyanitoff, R. G., and Wright, B. E. (1979) J. Bucteriol. 140, 1008-1012 16. Dimond, R. L.,and Loomis,W.F. (1976) J. Biol. Chem. 2 6 1 , 2680-2687 17. Dimond, R. L., Farnsworth, P. A., and Loomis, W. F.(1976) Deu. Biol. 50,169-181 18. Park, D. J. M., and Wright, B. E. (1973) Comp. Prog. Biomed. 3 , 10-26 19. Wright, B. E. (1960) Proc. Natl. Acud. Sci. U. S. A. 4 6 , 798-803

Model of TricarboxylicAcid Cycle in Dictyostelium

SUPPLEMENTAL MATERIAL TO: SYSTEMS ANALYSIS OF THE TRICARBOXYLIC ACID CYCLE IN DlrlYOSlELIUM DlSCOlDEUM

3105

I.

THE BASIS FOR HOOEL CONSTRUCTION H. B u t l e r and K a t h y R . Albe TABLE 4 RATE' EQUATIONS USED I N TCA MIDEL

Barbara E. W i g h t ,M a r g a r e t TABLE 1 ENZYME MECHANISMS AND KINETIC CONSTANTS DETERWINED LH KLEQ ENZYME REACTION 1 2

K. (nM) GluDH
2KG 2-KGDC NAD CoA Glu-2.0 NAD = 0.2

K,

(mnl

MECHANISH REFERENCE ENZYME

--

NH,=3.0 NAOH 0.025

1.0 0.07 0.002

NAOH 0.018 SucCoA 0.004 Fum = 0.4 O M 0.003 NAD NADH

0 04

--

Rapid E q u i l l b rO i urm dered Bi Bi

M u lP tir ni g te w i[tP hI
U"l u n i

50

Pong

41

U n i n o l e c u l a r Mars A c t i o n Rate K[A] Aspartate -,Oxaloacetate I (18): ENZYMES MODELED: C I t r a t e 1 -> I s o c ? t r a t e (12)': O x a l o a c e t a t e 1 -> A s p a r t a t e (20); A l a n i n e - > Pyruvate ( 7 ) : Glutamate ~> s u c c i n a t e 1 ( 1 4 ) ; Succinate 1 - > Glutamate ( 1 9 ) : O x a l o a c e t a t e 1 - > O x a l o a c e t a t e 2 (17):Aspartate -> O x a l o a c e t a t e2 ( I O ) ; O x a l o a c e t a t e2 - > A s p a r t a t e ( 9 ) . Dead-end C o r n p e t l t l v e l n h i b l t l o n P l n g Pong B i B i Rate VI[A][B)/(KB[A]+WI(l.O+[~NHl/K~)[B~+[A][B]) ENZYMES MODELED: Glutamate Dehydrogenase ( I ) ; C l t r s t e Synthase (11); I P O C i t l a t e Dehydrogenase (13). A l l o r t e n cM i c h a e l l r - M e n t e n Rate Vl[Al[E]/(WI+[Al)(KEPi[E]) ENZYMES MODELED: M a l i c Enzyme ( 6 ) .

3
5

SOH

Suc

- 0.22' --0 0.01 .37

MDH

Hal = 1.33 NAD = 0.10 O M 0.27 Mal NADP

--0.31

42
30

Iro Ordered BI B i

6 8

ME
PDC

A l l o s t eM r iic chaellr-Menten NADH = 0.049 ACO 0.024 [PI

39
21

Pyr 0.14 CoA 0.OOB NAD = 0.11 O Rapid M = 0.007 ACO = 0 01

--

M u l tP i ri in te g 0

u n iu o i Rate V I x V2([Al-[Pl/KEP)/(KP x V2 + V2[AI + VI[PI/KEP) Succinate Oehydrogenare (3); Fumarase ( 4 ) . ENZYHES MOOELED:

Pong w i t h

I1
13

CS

Equilibrium Ordered
CoA Bi = Bi 0.11

43 40

IracDH

lrac 0 13 NADH = 0 34
Asp ZKG
2KG

I s m = 0.13 NAD 0.34 NAOH = 0.02

RE aq pm i dl l bO r lrudm ered BB i i
Ping Pong
81 B i

I5

AlpTA AlaTA

16Ala

0.46 0.33 0.43 0.19

48' 48' KP x KIP) ENZYMES MODELED:

Pi"" ....,
D"""

P i n g Pang B1 81

Malate Dehydrogenase

(5)

n,

' O r l g i n l l e s t i m a t e of K . was 0.1 mfl Which was used i n t h e a n a l y z e d m a d e l . ' S e e a l s o : Komrnunlecki. P R ( 1 9 7 7 ) Ph.0. D l r r e r t a t i a n , Univelslty of MdslaChuIetts at Amherrt. LEGEND: A b b r e v i a t l a n r GluDH glutamate dehydrogenase, Z~KGDC: 2 - k e t o g l u t a r a t e dehydrogenase; ME: m 1 1 c enzyme; MOH: m a l a t e dehydrogenase complex; SOH: succinate dehydrogenase; PDC. pyruvate dehydrogenase complex; CS:cltrate Synthase; IrocOH: 1lOCltrdte dehydrogenase, AlaTA: a l a n ~ n e t r m s m l n a i e : ArpTA. a s p a r t a t e tranpamlnlse.

Rate = V I X vZ([A][B] - [PI[PI/KEP)/DENOM KB x V2[A1 + WI x VZ I B ) + V2[Al[B] + KP x VI[PI/KEQ + KP x where OENOM VI[PI/KEP + VI[PI[PI/KEP + KP x V I I A I [ P I / ( K I A x KEPI + WI x V2[BI[Pl/KIP ENZYHES MOOELED: Aspartate Transaminare ( I S ) ; Alanine Transaninare (16).

CI.

' Taken from Segel, I.H. (1975) Enzyme K l n e t i c r :B e h a v i o r and a n a l y s i s o f r a p r de q u i l i b r i u m and r t e a d y ~ r t a t eenzyme r y i t e m r . John Y l l e y 8 Sans, New York.

' Numbers ~n parentheses refer t or e a c t i o n si n ' KZ = K I B x KIC x KP/(KR x KIP)

Flgure I .

TABLE 5: FLUXES REACTION'

NET REACTION RATES DETERMINED FROM TRACER EXPERIMENTS COHPAREO TO STEADY-STATE METASIH MODEL

TFLUX Model'
2.40 2.85 2.60 1.90 1.40 I.90

Asp - > 0111

I4 I8

+ +

2.10
19 20 0 0

METASIM MOOEL InH/Mi"l 0.15 2.41 2.77 2.85 1.76 1.09 2.00 2.00 0 0

' H e t a b o l l t ep o o ld e r r g n a t l o n ra r eC o n l i f t e n tw t h Flgure 1. ' see reference 37; valuer bared on i S I v m e dm l t o c h o n d r l a l volume. TABLE 6: FLUX AND METABOLITE CONCENTRATIONS I N STEADY-STATE MODEL AT FIVE MINUTES

' M e t a b a l l t ec o n c e n t r a t l o n r are t h o s e d s t e r m n e d b y K e l l y .e t t o c e l l volume r a t l o o f 1 5 p o o l ra r r u m i n g a m..ochandria

a 1 . (36, 3 7 ) far m l t o c h o n d r l a l