Adenine and adenosine salvage pathways in erythrocytes and the role of S-adenosylhomocysteine hydrolase A theoretical study using elementary flux modes Stefan Schuster and Dimitar Kenanov Department of Bioinformatics, Friedrich Schiller University, Jena, Germany The human erythrocyte has been a subject not only of intense experimental research but also of many model- ling studies [1–6] because this cell is of high medical relevance, is readily accessible and its metabolism is relatively simple. Human red blood cells are not able to synthesize ATP de novo. However, they involve sal- vage pathways, that is, routes by which nucleosides or bases can be recycled to give nucleotide triphosphates [7]. The exact structure of salvage pathways (for exam- ple, starting from adenine or adenosine) has not yet been analysed in much detail. Because the salvage pathways involve enzymes consuming ATP, such as phosphoribosylpyrophosphate synthetase and adeno- sine kinase, as well as enzymes producing ATP, such as pyruvate kinase, it is not straightforward to see whether a net production of ATP can be realized. Besides adenine and adenosine, hypoxanthine is usu- ally considered a major substrate of salvage pathways [7]. However, in mature erythrocytes, hypoxanthine cannot be recycled to give ATP because of the lack of adenylosuccinate synthetase, which is necessary for transforming inosine 5¢-monophosphate (IMP) into AMP [8]. Here, we analyse theoretically how many sal- vage pathways exist, which enzymes each of these involves and in what flux proportions (i.e. relative fluxes) the enzymes operate. Moreover, we compute the net overall stoichiometry of ATP anabolism. (Throughout the paper, by ATP anabolism or buildup, Keywords elementary flux modes; enzyme deficiencies; erythrocytes; nucleotide metabolism; salvage pathways Correspondence S. Schuster, Department of Bioinformatics, Friedrich Schiller University, Ernst-Abbe- Platz 2, 07743 Jena, Germany Fax: +49 3641 946452 Tel: +49 3641 949580 E-mail: schuster@minet.uni-jena.de (Received 6 June 2005, revised 5 August 2005, accepted 19 August 2005) doi:10.1111/j.1742-4658.2005.04924.x This article is devoted to the study of redundancy and yield of salvage pathways in human erythrocytes. These cells are not able to synthesize ATP de novo. However, the salvage (recycling) of certain nucleosides or bases to give nucleotide triphosphates is operative. As the salvage pathways use enzymes consuming ATP as well as enzymes producing ATP, it is not easy to see whether a net synthesis of ATP is possible. As for pathways using adenosine, a straightforward assumption is that these pathways start with adenosine kinase. However, a pathway bypassing this enzyme and using S-adenosylhomocysteine hydrolase instead was reported. So far, this route has not been analysed in detail. Using the concept of elementary flux modes, we investigate theoretically which salvage pathways exist in erythro- cytes, which enzymes belong to each of these and what relative fluxes these enzymes carry. Here, we compute the net overall stoichiometry of ATP build-up from the recycled substrates and show that the network has con- siderable redundancy. For example, four different pathways of adenine sal- vage and 12 different pathways of adenosine salvage are obtained. They give different ATP ⁄ glucose yields, the highest being 3 : 10 for adenine sal- vage and 2 : 3 for adenosine salvage provided that adenosine is not used as an energy source. Implications for enzyme deficiencies are discussed. Abbreviations ADPRT, adenine phosphoribosyltransferase; IMP, inosine 5¢-monophosphate; SAHH, S-adenosylhomocysteine hydrolase; SAM, S-adenosylmethionine. 5278 FEBS Journal 272 (2005) 5278–5290 ª 2005 FEBS we mean the production of ATP from salvaged sub- strates rather than de novo synthesis.) As for pathways involving adenosine, a plausible assumption is that adenosine kinase would be used. However, Simmonds and coworkers [8–11] found that an elevation of ATP can occur in the absence of adenosine kinase, as long as adenine phosphoribosyl transferase (ADPR transferase, or ADPRT) is present. This is indicative of an alternative salvage pathway in human erythrocytes, and evidence was presented [8–11] that S-adenosylhomocysteine hydrolase (SAHH, EC 3.3.1.1), which is difficult to assess in vivo, is involved in these pathways. Since adenine is a substrate of ADPRT, the elevation of ATP in the absence of adenosine kinase shows that adenine must be released in the process before being incorporated into ATP. Indeed, studies on purified SAHH showed that several purine nucleosides and analogues can release adenine resulting from interaction with this enzyme [12]. One of these analogues is S-adenosylmethionine (SAM) [11] which can be taken up through the erythrocyte mem- brane and is abundant in all living cells [9,11]. Sim- monds and coworkers [8–11] investigated the pathway of ATP buildup from SAM, though not by a detailed stoichiometric analysis. SAM is converted into S-adenosylhomocysteine (the substrate of SAHH) by enzymes from the class of methyltransferases (EC 2.1.1.x). In the catalytic process of SAHH, addition- ally a spontaneous decomposition of the metabolite 3¢-ketoadenosine occurs, leading to free adenine and 3¢-ketoribose [13]. The adenine moiety can then be processed through ADPRT. Although under normal circumstances this pathway is not expected to produce significant amounts of adenine, it is important to men- tion the possibility this pathway offers not only for ATP generation (in erythrocytes or other types of cells harbouring SAHH) but also for the conversion of nucleoside analogues ⁄ derivatives to nucleotides. This is very important from the medical point of view because these analogues are used in chemotherapy, where one is interested in preventing an undesired transformation of these analogues [10]. Also in our present theoretical study, we include the enzyme SAHH and a methyl- transferase. Our analysis is based on the concept of ‘elementary flux mode’. This term refers to a minimal group of enzymes that can operate at steady state with all the irreversible reactions used in the right direction [14,15]. If only the enzymes belonging to one elementary mode are operative and, thereafter, one of the enzymes is inhibited, then the remaining enzymes can no longer be operational because the system cannot any longer main- tain a steady state. Elementary mode analysis has been applied to various systems (e.g [3,16–19]). C¸ akiy´ r et al. [6] applied this method to energy metabolism in erythro- cytes. A concept related to that of elementary modes is that of extreme pathways [20]. A comparison of the two concepts was made by Klamt and Stelling in [21]. Many biochemically relevant products are synthesized or degraded on multiple routes. Elementary modes pro- vide a powerful tool for determining the degree of multi- plicity and, thus, of redundancy [18,19]. This is of particular interest for the study of diseases based on enzyme deficiencies [3,6]. There are several diseases caused by enzyme deficiencies in nucleotide metabolism. Examples are provided by the following diseases: severe combined immunodeficiency, 2,8-dihydroxyadenine urolithiasis, and Lesch–Nyhan syndrome, caused by deficiencies in the adenosine deaminase (ADA), ADP- RT, and hypoxanthine guanine phosphoribosyltrans- ferase (HGPRT), respectively [22]. However, these diseases are related mainly to cells other than erythro- cytes, such as lymphocytes. In the case of severe deficiencies, a possible model- ling strategy is to consider the enzyme to be fully inhibited and examine which elementary modes are still present in the system. This allows us to detect bypas- ses, if any, or in other words to estimate the redund- ancy of the system. In this way one can predict which final products are still being produced and assess the impact of the deficiency on the patient’s metabolism. This, in turn, helps us decide which enzyme deficiencies can be considered as not harmful for the cell. Here, we specifically perform this analysis for ATP anabolism in erythrocytes. Results and Discussion As outlined in the Introduction, we compute element- ary flux modes in nucleotide metabolism. The reaction scheme is shown in Fig. 1. The scheme is explained in more detail in the Experimental procedures. The goal is to analyse the redundancy and molar yields of sal- vage pathways. This analysis is carried out consecu- tively for different substrates. For the simulation of adenine and adenosine salvage, we do not include methyltransferase and SAHH. Adenine salvage In the first simulation, we consider, in addition to the external metabolites mentioned in Experimental proce- dures, adenine as external, to find out how ATP can be synthesized starting from adenine. Running meta- tool on this network gives 153 elementary modes (supplementary Table S1). Four of them produce ATP S. Schuster and D. Kenanov A theoretical study using elementary flux modes FEBS Journal 272 (2005) 5278–5290 ª 2005 FEBS 5279 (modes 136–139, supplementary Table S1). They are listed in Table 2. Note that in Tables 2–5, the numbers in the brackets denote relative fluxes carried by the corresponding enzymes. + and – indicate whether the elementary mode remains intact if the enzyme in the column heading is deficient. It can be seen that mode II.1 (here and in the follow- ing, mode x,y means mode y in Table x) uses glycolysis, the oxidative pentose phosphate pathway, and the enzymes d-ribose-5P-isomerase (R5PI), phosphoribosyl- pyrophosphate (PRPP) synthase, ADPRT and adenyl- ate kinase (ApK). Mode II.2 involves glycolysis, both the oxidative and nonoxidative parts of the pentose phosphate pathway, and the enzymes R5PI, PRPP syn- thase, ADPRT and ApK, yet in proportions different from mode II.1. It is worth noting that glucose-6P-iso- merase (PGI) is used backwards (in the direction of glucose-6-phosphate formation) and that fructose- diphosphate aldolase and triosephosphate isomerase (TPI) are not involved. Mode II.3 involves ALD and TPI in addition but not PGI (Table 2). As for mode II.4, it is worth noting that it does not comprise the oxi- dative pentose phosphate pathway. Fructose-diphos- phate aldolase, TPI as well as PGI are involved in that mode. Importantly, none of these pathways involves adenosine kinase (AK), nor do they run via adenosine. Part of the pentose phosphate pathway is needed to pro- vide the R5P necessary for the ribose moiety in the nucleotides. As mentioned in the Introduction, due to the exist- ence of both ATP consuming reactions and ATP pro- ducing reactions in the salvage pathways, it is not easy to see whether a net production of ATP is possible. Note that only a certain fraction of the ATP produced in the lower part of glycolysis is obtained in the net balance because the remaining fraction is needed to ‘upgrade’ adenine. Let us analyse, for example, mode II.1. Two moles of adenine are converted into two AMP by ADPRT. The supply of two PRPP for this conversion requires two ATP in PRPP synthase. eCLGtx CLG KH PTA PDA P6GFP6 IGP P6LG PDF AGP3 PAHD D LAHDPAG GPD3, 1 IPT DAN HDAN GP3 PDMG GPD3,2 PDG esa KGP mi C LG PTA PDA P6GDH esaLG P OG P 6 O C 2 U RP5 PDAN HSG2 GGSS HPDAN S GxoHGRGS S P5R P5X P7S AGP3 P6F P4E K T IT A IP5R E P5 u X KTII AGP3 GP2 MGP PD AP T A PEP NE KP PDA PTA RYP t xeRYP sn a r t RYP CAL CALetx C A Ltrasn HDL H DAN D AN N ED AI EN P PR P T RP DA PMA PMI ODA ON I PM ADA C UN ADA C UN X PYH PPRP TR P G H P1R X P YHe t x P5R MRP y sPPR Pn P T A PMA PTA KA P DAP D A PMA pAK esaPNP P TA P DA a N + aN + kaelaN K + K + k a e l K KaNa P T A s e enarbmem M AS tx e MAS 2 HHAS odA -S H y c TM Y C H 1HH AS o biRot eK '3 es cA c YCH FPK HD P6 LG XHart ns ccAt eM + + Fig. 1. Model representing glycolysis, the pentose phosphate pathway and purine metabolism in red blood cells, including a methyltrans- ferase and two possible ways of operation of S-adenosylhomocysteine hydrolase (SAHH1 and SAHH2) (extended from [10]). Transport reac- tions of adenine and adenosine across the cell membrane are not shown for simplicity’s sake. For abbreviations of enzymes and metabolites, see Table 1. A theoretical study using elementary flux modes S. Schuster and D. Kenanov 5280 FEBS Journal 272 (2005) 5278–5290 ª 2005 FEBS ADPR transferase and PRPP synthase together form four AMP. Using another four ATP, these are trans- formed into eight ADP in ApK. Due to the special flux distribution, seven ATP are consumed in hexo- kinase and five ATP in phosphofructokinase. In glyco- lysis, 20 mol ATP are produced; 10 in each of phosphoglycerate kinase and pyruvate kinase. This gives an ATP balance of )2–4)7–5+10+10 ¼ 2. Note that the lower part of glycolysis has to run five times as fast as ADPR transferase to make this positive bal- ance possible. The ATP ⁄ glucose yields (that is, the ratios of ATP production over glucose consumption fluxes) of modes II.1-II.4 are 2 : 7, 1 : 6, 1 : 4 and 3 : 10, respectively. Note that these are the yields for the buildup of ATP from adenine rather than from ADP as usually indicated for glycolysis. Mode II.4 has the highest yield. It can be shown that the flux distri- bution realizing the highest yield always coincides with an elementary mode or a linear combination of two modes with the same maximum yield [14]. Thus, there Table 1. List of all enzymes and metabolites included in the model. Abbreviation Full name EC number Enzyme ADA Adenosine deaminase 3.5.4.4 ADPRT Adenine phosphoribosyltransferase 2.4.2.7 AK Adenosine kinase 2.7.1.20 ALD1 Fructose-diphosphate aldolase 4.1.2.13 AMPDA Adenosine monophosphate deaminase 3.5.4.6 APK Adenylate kinase 2.7.4.3 C5MT Cytosine-5-methyltransferase 2.1.1.37 DPGase Diphosphoglycerate phosphatase 3.1.3.13 DPGM 2,3-Diphosphoglycerate mutase 5.4.2.4 EN Enolase 4.2.1.11 G6PDH Glucose-6P dehydrogenase 1.1.1.49 GAPDH Glyceraldehyde-3P dehydrogenase 1.2.1.12 GL6PDH 6P-Gluconate dehydrogenase 1.1.1.49 GSHox Glutathioneperoxidase 1.11.1.9 GSSGR Glutathione reductase 1.8.1.7 HGPRT Hypoxanthine guanine phosphoribosyltransferase 2.4.2.8 HK Hexokinase 2.7.1.1 LDH Lactate dehydrogenase 1.1.1.27 NUC AMP phosphatase 3.1.3.5 PFK1 Phosphofructokinase 2.7.1.11 PGI Glucose-6P-isomerase 5.3.1.9 PGK1 Phosphoglycerate kinase 1 2.7.2.3 PGLase 6P-Gluconolactonase 3.1.1.31 PGM Phosphoglycerate mutase 1 5.4.2.1 PK Pyruvate kinase 2.7.1.40 PNPase Purine nucleoside phosphorylase 2.4.2.1 PRM Phosphoribomutase 5.4.2.7 PRPP synthase Phosphoribosylpyrophosphate synthetase 2.7.6.1 R5PI D-Ribose-5P-isomerase 5.3.1.6 SAHH S-Adenosylhomocysteine hydrolase 3.3.1.1 TA Transaldolase 2.2.1.2 TK Transketolase 2.2.1.1 TPI Triosephosphate isomerase 1 5.3.1.1 XU5PE D-Xylulose-5P-3-epimerase 5.1.3.1 Metabolites 1,3 DPG 1,3-Diphospho- D-glycerate 2,3 DPG 2,3-Diphospho- D-glycerate 2PG 2-Phospho- D-glycerate 3¢-keto ribose 3¢-Keto ribose 3PG 3-Phospho- D-glycerate Acc Acceptor for methyl group Adenine Adenine Ado Adenosine ADP Adenosine 5¢-diphosphate AMP Adenosine 5¢-monophosphate ATP Adenosine 5¢-triphosphate CO2 Carbon dioxide DHAP Dihydroxyacetone phosphate E4P D-Erythrose 4-phosphate F6P Fructose 6-phosphate FDP Fructose 1,6-diphosphate G6P Glucose 6-phosphate Table 1. Continued. Abbreviation Full name EC number GA3P Glyceraldehyde 3-phosphate GL6P D-Glucono-1,5-lactone 6-phosphate GLC Glucose GO6P 6-Phospho- D-gluconate GSH Reduced glutathione GSSG Oxidized glutathione HCY L-Homocysteine HYPX Hypoxanthine IMP Inosine 5¢-monophosphate INO Inosine K + Potassium LAC L-Lactate MetAcc Methylated acceptor Na + Sodium NAD Nicotinamide adenine dinucleotide NADH Nicotinamide adenine dinucleotide reduced NADP Nicotinamide adenine dinucleotide phosphate NADPH Nicotinamide adenine dinucleotide phosphate reduced PEP Phosphoenolpyruvate PRPP 5-Phospho-alpha- D-ribose 1-diphosphate PYR Pyruvate R5P D-Ribulose 5-phosphate RIP D-Ribose 1-phosphate RU5P D-Ribulose 5-phosphate S-AdoHcy S-Adenosyl- L-homocysteine S7P D-Sedoheptulose 7-phosphate SAM S-Adenosyl- L-methionine X5P D-Xylulose 5-phosphate S. Schuster and D. Kenanov A theoretical study using elementary flux modes FEBS Journal 272 (2005) 5278–5290 ª 2005 FEBS 5281 can be no flux distribution of adenine salvage enabling an ATP ⁄ glucose yield higher than 0.3. Interestingly, none of the ATP producing modes involves the 2,3-diphosphoglycerate phosphatase (DPG) bypass. As this would circumvent the enzyme phosphoglycerate kinase, the ATP yield of glycolysis would be decreased, to such an extent that no ATP buildup from adenine would be possible. Most of the remaining elementary modes of the first simulation can be interpreted as degradation of ATP to hypoxanthine. One elementary mode describes the 2,3DPG bypass of glycolysis, with a zero ATP balance. As we consider ADP as internal, normal glycolysis implying a transformation of ADP into ATP is not computed. Adenosine salvage In the second simulation, we analysed ATP buildup from adenosine. Therefore, we consider adenosine (but not adenine) to be external. This gives rise to 97 ele- mentary modes (Supplementary Table S2). Twelve modes (numbers 10, 15, 20, 54–59, 77, 85, and 92 in Table S2) produce ATP from adenosine (Table 3). All of these involve AK and ApK. Mode III.1 is made up of glycolysis, AK and ApK and does not involve any pentose phosphate pathway enzyme. The flux ratio between the upper and lower parts of glycolysis is, as in pure glycolysis, 1 : 2. The flux ratio between AK as well as ApK and the upper part of glycolysis is 2 : 3. Thus, 2 out of six ATP pro- duced from ADP in glycolysis are used to convert adenosine into AMP. The latter is ‘upgraded’ by ApK to give ADP. In total, 2 mol of ATP are built up from adenosine per 3 mol of glucose. Modes III.2 and III.3 involve different combinations of glycolysis and the pentose phosphate pathway as well as AK and ApK. The involvement of the pentose phosphate pathway is not, however, essential for ATP build up in these modes. It merely lowers the ATP ⁄ glucose yield. Modes III.4-III.9 do not start from glucose but solely from adenosine. This is used not only as the source for ATP buildup but also as an energy source. Adenosine is degraded into hypoxanthine (which is excreted) and ribose-1-phosphate, which is trans- formed, by the pentose phosphate pathway, into glyco- lytic intermediates. Modes III.10-III.12 use both glucose and adenosine as energy sources, in different proportions. Modes III.4, III.7 and III.11 involve the 2,3DPG bypass. Again, there is no mode involving the 2,3DPG bypass when glucose is used as the only energy source (modes III.1-III.3) because the ATP ⁄ glu- cose yield would then be so low that no ATP buildup would be possible. The ATP ⁄ adenosine yields of the ATP-producing modes are 1 for modes III.1-III.3, 1 : 4, 2 : 5, 1 : 4, 1 : 4, 8 : 17, 5 : 14, 2 : 3, 1 : 4 and 5 : 8 for modes III.4-III.12, respectively. Thus, modes starting from glucose and adenosine transform the lat- ter completely into ATP, which implies that glucose is the only energy source. By contrast, in the modes starting solely from adenosine, part of this substrate is used as an energy source, so that the yield is lower. Inclusion of SAHH As mentioned in the Introduction, there is experimen- tal evidence that S-adenosylmethionine can be used by erythrocytes for ATP buildup [8–11]. To analyse this Table 2. Elementary modes producing ATP from adenine. Elementary modes –ADA –AK –PNPase –ADPRT 1. (5 PGI) (5 ALD) (5 TPI) (10 GAPDH) (10 PGM) (10 EN) (10 LACex) (– 4 ApK) (2 PGLase) (4 GSSGR) (2 R5PI) (7 HK) (5 PFK) (10 PGK) (10 PK) (10 LDH) (2 ADPRT) (4 GSHox) (2 PRPPsyn) (2 G6PD) (2 GL6PDH) 7 GLC + 2 Adenine ¼ 2CO 2 + 10 LACext + 2 ATP +++ – 2. ()10 PGI) (5 GAPDH) (5 PGM) (5 EN) (5 LACex) () 2 ApK) (16 PGLase) (32 GSSGR) (6 R5PI) (10 Xu5PE) (5 TKI) (5 TKII) (5 TA) (6 HK) (5 PGK) (5 PK) (5 LDH) ADPRT (32 GSHox) PRPPsyn (16 G6PD) (16 GL6PDH) 6 GLC + Adenine ¼ 16 CO 2 + 5 LACext + ATP +++ – 3. (2 ALD) (2 TPI) (5 GAPDH) (5 PGM) (5 EN) (5 LACex) () 2 ApK) (4 PGLase) (8 GSSGR) (2 R5PI) (2 Xu5PE) TKI TKII TA (4 HK) (2 PFK) (5 PGK) (5 PK) (5 LDH) ADPRT (8 GSHox) PRPPsyn (4 G6PD) (4 GL6PDH) 4 GLC + Adenine ¼ 4CO 2 + 5 LACext + ATP +++ – 4. (10 PGI) (8 ALD) (8 TPI) (15 GAPDH) (15 PGM) (15 EN) (15 LACex) (– 6 ApK) (2 R5PI) (– 2 Xu5PE) -TKI -TKII -TA (10 HK) (8 PFK) (15 PGK) (15 PK) (15 LDH) (3 ADPRT) (3 PRPPsyn) 10 GLC + 3 Adenine ¼ 15 LACext + 3 ATP +++ – A theoretical study using elementary flux modes S. Schuster and D. Kenanov 5282 FEBS Journal 272 (2005) 5278–5290 ª 2005 FEBS in detail, we performed a simulation with the complete scheme shown in Fig. 1; that is, including at least one methyltransferase (considered irreversible in the direc- tion of S-adenosylmethionine consumption) and SAHH. In that simulation, adenine and adenosine were considered internal, while S-adenosylmethionine was treated as external. This gave rise to 214 element- ary modes (Supplementary Table S3). Twenty-three modes produce ATP (Table 4). Some of them involve the modes starting from adenine obtained in the first simulation and include methyltransferase and SAHH2 in addition. Some others involve the modes starting from adenosine obtained in the second simulation and include methyltransferases and SAHH1 in addition. Interestingly, some modes involve both SAHH1 and SAHH2. Table 3. Elementary modes producing ATP from adenosine. Elementary modes –ADA –AK –PNPase –ADPRT 1. (3 PGI) (3 ALD) (3 TPI) (6 GAPDH) (6 PGM) (6 EN) (6 LACex) ()2 ApK) (3 HK) (3 PFK) (6 PGK) (6 PK) (6 LDH) (2 AK) 3 GLC +2 ADO ¼ 6 LACext + 2 ATP +–+ + 2. ()6 PGI) (3 GAPDH) (3 PGM) (3 EN) (3 LACex) –ApK (9 PGLase) (18 GSSGR) (3 R5PI) (6 Xu5PE) (3 TKI) (3 TKII) (3 TA) (3 HK) (3 PGK) (3 PK) (3 LDH) (18 GSHox) (9 G6PD) (9 GL6PDH) AK 3 GLC + ADO ¼ 9CO 2 + 3 LACext + ATP +–+ + 3. (6 ALD) (6 TPI) (15 GAPDH) (15 PGM) (15 EN) (15 LACex) (–ApK) (9 PGLase) (18 GSSGR) (3 R5PI) (6 Xu5PE) (3 TKI) (3 TKII) (3 TA) (9 HK) (6 PFK) (15 PGK) (15 PK) (15 LDH) (18 GSHox) (9 G6PD) (9 GL6PDH) (5 AK) 9 GLC +5 ADO ¼ 9CO 2 + 15 LACext + 5 ATP +–+ + 4. (– 6 PGI) (3 GAPDH) (3 DPGM) (3 PGM) (3 EN) (3 LACex) –ApK (6 PGLase) (12 GSSGR) (6 Xu5PE) (3 TKI) (3 TKII) (3 TA) (3PNPase) (3 PRM) (3 HXtrans) (3 DPGase) (3 PK) (3 LDH) (12 GSHox) (3 ADA) (6 G6PD) (6 GL6PDH) AK 4 ADO ¼ 3 HYPXext + 6 CO 2 + 3LACext + ATP ––– + 5. ()6 PGI) (3 GAPDH) (3 PGM) (3 EN) (3 LACex) ()2 ApK) (6 PGLase) (12 GSSGR) (6 Xu5PE) (3 TKI) (3 TKII) (3 TA) (3 PNPase) (3 PRM) (3 HXtrans) (3 PGK) (3 PK) (3 LDH) (12 GSHox) (3 ADA) (6 G6PD) (6 GL6PDH) (2 AK) 5 ADO ¼ 3 HYPXext + 6 CO 2 + 3 LACext + 2 ATP ––– + 6. ()6 PGI) (3 GAPDH) (3 PGM) (3 EN) (3 LACex) –ApK (6 PGLase) (12 GSSGR) (6 Xu5PE) (3 TKI) (3 TKII) (3 TA) (3 PNPase) (3 PRM) (3 HXtrans) (3 PGK) (3 PK) (3 LDH) (3 AMPDA) (12 GSHox) (3 IMPase) (6 G6PD) (6 GL6PDH) (4 AK) 4 ADO ¼ 3 HYPXext + 6 CO 2 + 3 LACext + ATP +–– + 7. (2 ALD) (2 TPI) (5 GAPDH) (5 DPGM) (5 PGM) (5 EN) (5 LACex) –ApK ()2 R5PI) (2 Xu5PE) TKI TKII TA (3 PNPase) (3 PRM) (3 HXtrans) (2 PFK) (5 DPGase) (5 PK) (5 LDH) (3 ADA) AK 4 ADO ¼ 3 HYPXext +5 LACext + ATP ––– + 8. (6 ALD) (6 TPI) (15 GAPDH) (15 PGM) (15 EN) (15 LACex) ()8 ApK) ()6 R5PI) (6 Xu5PE) (3 TKI) (3 TKII) (3 TA) (9 PNPase) (9 PRM) (9 HXtrans) (6 PFK) (15 PGK) (15 PK) (15 LDH) (9 ADA) (8 AK) 17 ADO ¼ 9 HYPXext + 15 LACext + 8 ATP ––– + 9. (6 ALD) (6 TPI) (15 GAPDH) (15 PGM) (15 EN) (15 LACex) ()5 ApK) ()6 R5PI) (6 Xu5PE) (3 TKI) (3 TKII) (3 TA) (9 PNPase) (9 PRM) (9 HXtrans) (6 PFK) (15 PGK) (15 PK) (15 LDH) (9 AMPDA) (9 IMPase) (14 AK) 14 ADO ¼ 9 HYPXext + 15 LACext + 5 ATP +–– + 10. (2 ALD) (2 TPI) (5 GAPDH) (5 PGM) (5 EN) (5 LACex) ()2 ApK) (2 PGLase) (4 GSSGR) (2 Xu5PE) TKI TKII TA PNPase PRM HXtrans (2 HK) (2 PFK) (5 PGK) (5 PK) (5 LDH) (4 GSHox) ADA (2 G6PD) (2 GL6PDH) (2 AK) 2 GLC + 3 ADO ¼ HYPXext + 2 CO 2 + 5 LACext + 2 ATP ––– + 11. (6 ALD) (6 TPI) (15 GAPDH) (15 DPGM) (15 PGM) (15 EN) (15 LACex) –ApK (6 PGLase) (12 GSSGR) (6 Xu5PE) (3 TKI) (3 TKII) (3 TA) (3 PNPase) (3 PRM) (3 HXtrans) (6 HK) (6 PFK) (15 DPGase) (15 PK) (15 LDH) (12 GSHox) (3 ADA) (6 G6PD) (6 GL6PDH) AK 6 GLC + 4 ADO ¼ 3 HYPXext + 6 CO 2 + 15 LACext + ATP ––– + 12. (6 ALD) (6 TPI) (15 GAPDH) (15 PGM) (15 EN) (15 LACex) (–ApK) (6 PGLase) (12 GSSGR) (6 Xu5PE) (3 TKI) (3 TKII) (3 TA) (3 PNPase) (3 PRM) (3 HXtrans) (6 HK) (6 PFK) (15 PGK) (15 PK) (15 LDH) (3 AMPDA) (12 GSHox) (3 IMPase) (6 G6PD) (6 GL6PDH) (8 AK) 6 GLC + 8 ADO ¼ 3 HYPXext + 6 CO 2 + 15 LACext + 5 ATP +–– + S. Schuster and D. Kenanov A theoretical study using elementary flux modes FEBS Journal 272 (2005) 5278–5290 ª 2005 FEBS 5283 Table 4. ATP producing modes in the extended system including SAHH and methyltransferase. Elementary modes –ADA –AK –PNPase –ADPRT Through SAHH1 but not SAHH2 1. (3 DPGase) (3 PK) (3 LDH) (4 MT) (12 GSHox) (3 ADA) (6 G6PD) (6 GL6PDH) AK (– 6 PGI) (3 GAPDH) (3 DPGM) (3 PGM) (3 EN) (3 LACex) -ApK (6 PGLase) (12 GSSGR) (6 Xu5PE) (3 TKI) (3 TKII) (3 TA) (3 PNPase) (3 PRM) (3 HXtrans) (4 SAHH1) 4 SAM + 4 H 2 O+4Acc¼ 3 HYPXext + 6 CO 2 + 4 HCY + ATP + 3 LACext + 4 MetAcc ––– + 2. (3 PGK) (3 PK) (3 LDH) (5 MT) (12 GSHox) (3 ADA) (6 G6PD) (6 GL6PDH) (2 AK) ()6 PGI) (3 GAPDH) (3 PGM) (3 EN) (3 LACex) ()2 ApK) (6 PGLase) (12 GSSGR) (6 Xu5PE) (3 TKI) (3 TKII) (3 TA) (3 PNPase) (3 PRM) (3 HXtrans) (5 SAHH1) 5 SAM + 5 H 2 O+5Acc¼ 3 HYPXext + 6 CO 2 +5 HCY + 2 ATP + 3 LACext + 5 AccMet ––– + 3. (3 PGK) (3 PK) (3 LDH) (3 AMPDA) (4 MT) (12 GSHox) (3 IMPase) (6 G6PD) (6 GL6PDH) (4 AK) ()6 PGI) (3 GAPDH) (3 PGM) (3 EN) (3 LACex) –ApK (6 PGLase) (12 GSSGR) (6 Xu5PE) (3 TKI) (3 TKII) (3 TA) (3 PNPase) (3 PRM) (3 HXtrans) (4 SAHH1) 4 SAM + 4 H 2 O+4Acc¼ 3 HYPXext + 6 CO 2 + 4 HCY + ATP + 3 LACext + 4 AccMet +–– + 4. (2 PFK) (5 DPGase) (5 PK) (5 LDH) (4 MT) (3 ADA) AK (2 ALD) (2 TPI) (5 GAPDH) (5 DPGM) (5 PGM) (5 EN) (5 LACex) –ApK ()2 R5PI) (2 Xu5PE) TKI TKII TA (3 PNPase) (3 PRM) (3 HXtrans) (4 SAHH1) 4 SAM +4 H 2 O+4Acc¼ 3 HYPXext + 4 HCY + ATP + 5 LACext + 4 AccMet ––– + 5. (6 PFK) (15 PGK) (15 PK) (15 LDH) (17 MT) (9 ADA) (8 AK) (6 ALD) (6 TPI) (15 GAPDH) (15 PGM) (15 EN) (15 LACex) ()8 ApK) ()6 R5PI) (6 Xu5PE) (3 TKI) (3 TKII) (3 TA) (9 PNPase) (9 PRM) (9 HXtrans) (17 SAHH1) 17 SAM +17 H 2 O +17 Acc ¼ 9 HYPXext + 17 HCY + 8 ATP + 15 LACext +17 AccMet ––– + 6. (6 PFK) (15 PGK) (15 PK) (15 LDH) (9 AMPDA) (14 MT) (9 IMPase) (14 AK) (6 ALD) (6 TPI) (15 GAPDH) (15 PGM) (15 EN) (15 LACex) ()5 ApK) ()6 R5PI) (6 Xu5PE) (3 TKI) (3 TKII) (3 TA) (9 PNPase) (9 PRM) (9 HXtrans) (14 SAHH1) 14 SAM +14 H 2 O + 14 Acc ¼ 9 HYPXext +14 HCY + 5 ATP + 15 LACext + 14 AccMet +–– + 7. (3 HK) (3 PGK) (3 PK) (3 LDH) MT (18 GSHox) (9 G6PD) (9 GL6PDH) AK ()6 PGI) (3 GAPDH) (3 PGM) (3 EN) (3 LACex) –ApK (9 PGLase) (18 GSSGR) (3 R5PI) (6 Xu5PE) (3 TKI) (3 TKII) (3 TA) SAHH1 SAM + H 2 O + Acc +3 GLC ¼ 9CO 2 + HCY + ATP +3 LACext + AccMet +–+ + 8. (6 HK) (6 PFK) (15 DPGase) (15 PK) (15 LDH) (4 MT) (12 GSHox) (3 ADA) (6 G6PD) (6 GL6PDH) AK (6 ALD) (6 TPI) (15 GAPDH) (15 DPGM) (15 PGM) (15 EN) (15 LACex) –ApK (6 PGLase) (12 GSSGR) (6 Xu5PE) (3 TKI) (3 TKII) (3 TA) (3 PNPase) (3 PRM) (3 HXtrans) (4 SAHH1) 4 SAM + 4 H 2 O + 4 Acc + 6 GLC ¼ 3 HYPXext + 6 CO 2 + 4 HCY + ATP + 15 LACext + 4 AccMet ––– + 9. (3 HK) (3 PFK) (6 PGK) (6 PK) (6 LDH) (2 MT) (2 AK) (3 PGI) (3 ALD) (3 TPI) (6 GAPDH) (6 PGM) (6 EN) (6 LACex) ()2 ApK) (2 SAHH1) 2 SAM +2 H 2 O + 2 Acc + 3 GLC ¼ 2 HCY + 2 ATP + 6 LACext + 2 AccMet +–+ + 10. (9 HK) (6 PFK) (15 PGK) (15 PK) (15 LDH) (5 MT) (18 GSHox) (9 G6PD) (9 GL6PDH) (5 AK) (6 ALD) (6 TPI) (15 GAPDH) (15 PGM) (15 EN) (15 LACex) (– 5 ApK) (9 PGLase) (18 GSSGR) (3 R5PI) (6 Xu5PE) (3 TKI) (3 TKII) (3 TA) (5 SAHH1) 5 SAM +5 H2O +5 Acc +9 GLC ¼ 9 CO2 +5 HCY +5 ATP +15 LACext +5 AccMet +–+ + 11. (2 HK) (2 PFK) (5 PGK) (5 PK) (5 LDH) (3 MT) (4 GSHox) ADA (2 G6PD) (2 GL6PDH) (2 AK) (2 ALD) (2 TPI) (5 GAPDH) (5 PGM) (5 EN) (5 LACex) ()2 ApK) (2 PGLase) (4 GSSGR) (2 Xu5PE) TKI TKII TA PNPase PRM HXtrans (3 SAHH1) 3 SAM +3 H 2 O + 3 Acc + 2 GLC ¼ HYPXext + 2 CO 2 + 3 HCY + 2 ATP + 5 LACext + 3 AccMet ––– + 12. (6 HK) (6 PFK) (15 PGK) (15 PK) (15 LDH) (3 AMPDA) (8 MT) (12 GSHox) (3 IMPase) (6 G6PD) (6 GL6PDH) (8 AK) (6 ALD) (6 TPI) (15 GAPDH) (15 PGM) (15 EN) (15 LACex) ()5 ApK) (6 PGLase) (12 GSSGR) (6 Xu5PE) (3 TKI) (3 TKII) (3 TA) (3 PNPase) (3 PRM) (3 HXtrans) (8 SAHH1) 8 SAM + 8 H 2 O + 8 Acc + 6 GLC ¼ 3 HYPXext + 6 CO 2 + 8 HCY + 5 ATP + 15 LACext + 8 AccMet +–– + Through SAHH1 & SAHH2 1. (4 DPGase) (4 PK) (4 LDH) (6 MT) ADPRT (16 GSHox) PRPPsyn (5 ADA) (8 G6PD) (8 GL6PDH)()8 PGI) (4 GAPDH) (4 DPGM) (4 PGM) (4 EN) (4 LACex) (– 2 ApK) (8 PGLase) (16 GSSGR) (8 Xu5PE) (4 TKI) (4 TKII) (4 TA) (5 PNPase) (5 PRM) (5 HXtrans) SAHH2 (5 SAHH1) 6 SAM + 6 H 2 O+6Acc¼ 5 HYPXext + 8 CO 2 +6 HCY + ATP + 4 LACext + 6 AccMet + 3KRibose –+– – A theoretical study using elementary flux modes S. Schuster and D. Kenanov 5284 FEBS Journal 272 (2005) 5278–5290 ª 2005 FEBS Note that operation of ATP-producing pathways starting from S-adenosylmethionine permanently util- izes a methyl acceptor and produces the corresponding methylated form. In our simulation, we consider both substances to be external. A more detailed model may include a regeneration of the methyl acceptor from the methylated form or from other sources. Another possi- bility is to consider the following reaction mechanism. As SAHH1 is reversible, adenosine may react with homocysteine halfway and then (via the SAHH2 func- tion) back to adenine, ribose and homocysteine. Thus, there is no net consumption of homocysteine in the process, and S-adenosylmethionine is not involved at all. Therefore, we performed a simulation with a model including the two functions of SAHH but excluding the methyltransferase (and, hence, S-adeno- sylmethionine). Adenosine was considered external. This produced 135 elementary modes (Supplementary Table 4. Continued. Elementary modes –ADA –AK –PNPase –ADPRT 2. (2 PGK) (2 PK) (2 LDH) (4 MT) ADPRT (8 GSHox) PRPPsyn (3 ADA) (4 G6PD) (4 GL6PDH) (– 4 PGI) (2 GAPDH) (2 PGM) (2 EN) (2 LACex) (– 2 ApK) (4 PGLase) (8 GSSGR) (4 Xu5PE) (2 TKI) (2 TKII) (2 TA) (3 PNPase) (3 PRM) (3 HXtrans) SAHH2 (3 SAHH1) 4 SAM + 4 H2O + 4 Acc ¼ 3 HYPXext + 4 CO2 +4 HCY + ATP + 2 LACext + 4 AccMet + 3KRibose –+– – 3. (8 PFK) (20 DPGase) (20 PK) (20 LDH) (18 MT) (3 ADPRT) (3 PRPPsyn) (15 ADA) (8 ALD) (8 TPI) (20 GAPDH) (20 DPGM) (20 PGM) (20 EN) (20 LACex) ()6 ApK) ()8 R5PI) (8 Xu5PE) (4 TKI) (4 TKII) (4 TA) (15 PNPase) (15 PRM) (15 HXtrans) (3 SAHH2) (15 SAHH1) 18 SAM + 18 H 2 O + 18 Acc ¼ 15 HYPXext + 18 HCY + 3 ATP + 20 LACext + 18 AccMet + 3 3KRibose –+– – 4. (2 PFK) (5 PGK) (5 PK) (5 LDH) (7 MT) (2 ADPRT) (2 PRPPsyn) (5 ADA) (2 ALD) (2 TPI) (5 GAPDH) (5 PGM) (5 EN) (5 LACex) ()4ApK)()2 R5PI) (2 Xu5PE) TKI TKII TA (5 PNPase) (5 PRM) (5 HXtrans) (2 SAHH2) (5 SAHH1) 7 SAM + 7 H 2 O+7Acc¼ 5 HYPXext + 7 HCY + 2 ATP + 5 LACext + 7 AccMet + 2 3KRibose –+– – 5. (8 HK) (8 PFK) (20 DPGase) (20 PK) (20 LDH) (6 MT) ADPRT (16 GSHox) PRPPsyn (5 ADA) (8 G6PD) (8 GL6PDH) (8 ALD) (8 TPI) (20 GAPDH) (20 DPGM) (20 PGM) (20 EN) (20 LACex) (– 2 ApK) (8 PGLase) (16 GSSGR) (8 Xu5PE) (4 TKI) (4 TKII) (4 TA) (5 PNPase) (5 PRM) (5 HXtrans) SAHH2 (5 SAHH1) 6 SAM + 6 H 2 O + 6 Acc + 8 GLC ¼ 5 HYPXext + 8 CO 2 + 6 HCY + ATP + 20 LACext + 6 AccMet + 3KRibose –+– – 6. (2 HK) (2 PFK) (4 PGK) (4 PK) (4 LDH) (2 MT) ADPRT PRPPsyn ADA (2 PGI) (2 ALD) (2 TPI) (4 GAPDH) (4 PGM) (4 EN) (4 LACex) ()2 ApK) PNPase PRM HXtrans SAHH2 SAHH1 2 SAM + 2 H 2 O + 2 Acc + 2 GLC ¼ HYPXext + 2 HCY + ATP + 4 LACext + 2 AccMet + 3KRibose –+– – 7. (4 HK) (4 PFK) (10 PGK) (10 PK) (10 LDH) (8 MT) (3 ADPRT) (8 GSHox) (3 PRPPsyn) (5 ADA) (4 G6PD) (4 GL6PDH) (4 ALD) (4 TPI) (10 GAPDH) (10 PGM) (10 EN) (10 LACex) ()6 ApK) (4 PGLase) (8 GSSGR) (4 Xu5PE) (2 TKI) (2 TKII) (2 TA) (5 PNPase) (5 PRM) (5 HXtrans) (3 SAHH2) (5 SAHH1) 8 SAM + 8 H 2 O + 8 Acc + 4 GLC ¼ 5 HYPXext + 4 CO 2 + 8 HCY + 3 ATP + 10 LACext + 8 AccMet + 3 3KRibose –+– – Through SAHH2 only 1. (5 PK) (5 LDH) MT ADPRT (32 GSHox) PRPPsyn (16 G6PD) (16 GL6PDH) ()10 PGI) (5 GAPDH) (5 PGM) (5 EN) (5 LACex) ()2 ApK) (16 PGLase) (32 GSSGR) (6 R5PI) (10 Xu5PE) (5 TKI) (5 TKII) (5 TA) SAHH2 SAM + H 2 O+Acc+6GLC¼ 16 CO 2 + HCY + ATP + 5 LACext + AccMet + 3KRibose +++ – 2. (10 HK) (8 PFK) (15 PGK) (15 PK) (15 LDH) (3 MT) (3 ADPRT) (3 PRPPsyn) (10 PGI) (8 ALD) (8 TPI) (15 GAPDH) (15 PGM) (15 EN) (15 LACex) ()6 ApK) (2 R5PI) ()2 Xu5PE) –TKI –TKII –TA (3 SAHH2) 3 SAM + 3 H 2 O + 3 Acc +10 GLC ¼ 3 HCY + 3 ATP + 15 LACext + 3 AccMet + 3 3KRibose +++ – 3. (4 HK) (2 PFK) (5 PGK) (5 PK) (5 LDH) MT ADPRT (8 GSHox) PRPPsyn (4 G6PD) (4 GL6PDH) (2 ALD) (2 TPI) (5 GAPDH) (5 PGM) (5 EN) (5 LACex) ()2 ApK) (4 PGLase) (8 GSSGR) (2 R5PI) (2 Xu5PE) TKI TKII TA SAHH2 SAM + H 2 O+Acc+4GLC¼ 4CO 2 + HCY + ATP + 5 LACext + AccMet + 3KRibose +++ – 4. (7 HK) (5 PFK) (10 PGK) (10 PK) (10 LDH) (2 MT) (2 ADPRT) (4 GSHox) (2 PRPPsyn) (2 G6PD) (2 GL6PDH) (5 PGI) (5 ALD) (5 TPI) (10 GAPDH) (10 PGM) (10 EN) (10 LACex) (– 4 ApK) (2 PGLase) (4 GSSGR) (2 R5PI) (2 SAHH2) 2 SAM + 2 H 2 O + 2 Acc + 7 GLC ¼ 2CO 2 + 2 HCY + 2 ATP + 10 LACext + 2 AccMet + 2 3KRibose +++ – S. Schuster and D. Kenanov A theoretical study using elementary flux modes FEBS Journal 272 (2005) 5278–5290 ª 2005 FEBS 5285 Table S4) of which 10 generate ATP from adenosine (Table 5). As expected, all of these use SAHH1 in the backward and SAHH2 in the forward direction. As can be seen in Table 5, both the ATP ⁄ glucose yield and ATP ⁄ adenosine yields are rather diverse. The highest values are 3 : 4 (in the modes really using glu- cose) and 1, respectively. However, they do not occur together, the elementary mode producing 3 mol of ATP from 4 mol of glucose requires 8 mol of adeno- sine. As for the modes allowing an ATP ⁄ adenosine yield of 1, the highest ATP ⁄ glucose yield is 3 : 10. It is worth noting that there are 14 more modes not including SAHH but producing ATP (Supplementary Table S4). Purine nucleoside phosphorylase, ADA, AK and ADPRT deficiencies By checking which of the computed elementary modes remain after deleting a given enzyme, it can easily be analysed which salvage pathways can be operative in spite of severe enzyme deficiencies. If ADA is deficient, Table 5. Elementary modes producing ATP in the presence of SAHH (but not methyltransferase). There are 14 more modes not including SAHH but producing ATP. Elementary modes –ADA –AK –PNPase –ADPRT 1. (5 PGI) (5 ALD) (5 TPI) (10 GAPDH) (10 PGM) (10 EN) (10 LACex) ()4 ApK) (2 PGLase) (4 GSSGR) (2 R5PI) ()2 SAHH1) (7 HK) (5 PFK) (10 PGK) (10 PK) (10 LDH) (2 ADPRT) (4 GSHox) (2 PRPPsyn) (2 G6PD) (2 GL6PDH) (2 SAHH2) 7 GLC + 2 ADO ¼ 2CO 2 +10 LACext + 2 3KRibose + 2 ATP +++ – 2. (10 PGI) (8 ALD) (8 TPI) (15 GAPDH) (15 PGM) (15 EN) (15 LACex) ()6 ApK) (2 R5PI) (– 2 Xu5PE) -TKI -TKII -TA ()3 SAHH1) (10 HK) (8 PFK) (15 PGK) (15 PK) (15 LDH) (3 ADPRT) (3 PRPPsyn) (3 SAHH2) 10 GLC + 3 ADO ¼ 15 LACext + 3 3KRibose + 3 ATP +++ – 3. ()10 PGI) (5 GAPDH) (5 PGM) (5 EN) (5 LACex) ()2 ApK) (16 PGLase) (32 GSSGR) (6 R5PI) (10 Xu5PE) (5 TKI) (5 TKII) (5 TA) -SAHH1 (6 HK) (5 PGK) (5 PK) (5 LDH) ADPRT (32 GSHox) PRPPsyn (16 G6PD) (16 GL6PDH) SAHH2 6 GLC + ADO ¼ 16 CO 2 +5 LACext + 3KRibose + ATP +++ – 4. ()8 PGI) (4 GAPDH) (4 DPGM) (4 PGM) (4 EN) (4 LACex) ()2 ApK) (8 PGLase) (16 GSSGR) (8 Xu5PE) (4 TKI) (4 TKII) (4 TA) (5 PNPase) (5 PRM) (5 HXtrans) )SAHH1 (4 DPGase) (4 PK) (4 LDH) ADPRT (16 GSHox) PRPPsyn (5 ADA) (8 G6PD) (8 GL6PDH) SAHH2 6 ADO ¼ 5 HYPXext + 8 CO 2 + 4 LACext + 3KRibose + ATP –+– – 5. ()4 PGI) (2 GAPDH) (2 PGM) (2 EN) (2 LACex) ()2 ApK) (4 PGLase) (8 GSSGR) (4 Xu5PE) (2 TKI) (2 TKII) (2 TA) (3 PNPase) (3 PRM) (3 HXtrans) –SAHH1 (2 PGK) (2 PK) (2 LDH) ADPRT (8 GSHox) PRPPsyn (3 ADA) (4 G6PD) (4 GL6PDH) SAHH2 4 ADO ¼ 3 HYPXext + 4 CO 2 + 2 LACext + 3KRibose + ATP –+– – 6. (8 ALD) (8 TPI) (20 GAPDH) (20 DPGM) (20 PGM) (20 EN) (20 LACex) ()6 ApK) ()8 R5PI) (8 Xu5PE) (4 TKI) (4 TKII) (4 TA) (15 PNPase) (15 PRM) (15 HXtrans) ()3 SAHH1) (8 PFK) (20 DPGase) (20 PK) (20 LDH) (3 ADPRT) (3 PRPPsyn) (15 ADA) (3 SAHH2) 18 ADO ¼ 15 HYPXext + 20 LACext + 3 3KRibose + 3 ATP –+– – 7. (2 ALD) (2 TPI) (5 GAPDH) (5 PGM) (5 EN) (5 LACex) ()4ApK)()2 R5PI) (2 Xu5PE) TKI TKII TA (5 PNPase) (5 PRM) (5 HXtrans) ()2 SAHH1) (2 PFK) (5 PGK) (5 PK) (5 LDH) (2 ADPRT) (2 PRPPsyn) (5 ADA) (2 SAHH2) 7 ADO ¼ 5 HYPXext + 5 LACext + 2 3KRibose + 2 ATP –+– – 8. (2 PGI) (2 ALD) (2 TPI) (4 GAPDH) (4 PGM) (4 EN) (4 LACex) ()2 ApK) PNPase PRM HXtrans -SAHH1 (2 HK) (2 PFK) (4 PGK) (4 PK) (4 LDH) ADPRT PRPPsyn ADA SAHH2 2 GLC + 2 ADO ¼ HYPXext + 4 LACext + 3KRibose + ATP –+– – 9. (4 ALD) (4 TPI) (10 GAPDH) (10 PGM) (10 EN) (10 LACex) (–ApK) (4 PGLase) (8 GSSGR) (4 Xu5PE) (2 TKI) (2 TKII) (2 TA) (5 PNPase) (5 PRM) (5 HXtrans) ()3 SAHH1) (4 HK) (4 PFK) (10 PGK) (10 PK) (10 LDH) (3 ADPRT) (8 GSHox) (3 PRPPsyn) (5 ADA) (4 G6PD) (4 GL6PDH) (3 SAHH2) 4 GLC + 8 ADO ¼ 5 HYPXext + 4 CO 2 + 10 LACext + 3 3KRibose + 3 ATP –+– – 10. (8 ALD) (8 TPI) (20 GAPDH) (20 DPGM) (20 PGM) (20 EN) (20 LACex) ()2 ApK) (8 PGLase) (16 GSSGR) (8 Xu5PE) (4 TKI) (4 TKII) (4 TA) (5 PNPase) (5 PRM) (5 HXtrans) –SAHH1 (8 HK) (8 PFK) (20 DPGase) (20 PK) (20 LDH) ADPRT (16 GSHox) PRPPsyn (5 ADA) (8 G6PD) (8 GL6PDH) SAHH2 8 GLC + 6 ADO ¼ 5 HYPXext + 8 CO 2 + 20 LACext + 3KRibose + ATP –+– – A theoretical study using elementary flux modes S. Schuster and D. Kenanov 5286 FEBS Journal 272 (2005) 5278–5290 ª 2005 FEBS all the four modes producing ATP from adenine remain intact because they do not involve ADA (Table 2). Out of the 12 modes producing ATP from adenosine, modes III.1-III.3, III.6, III.9, and III.12 remain intact. It is interesting that the other ATP-pro- ducing modes (which drop out) involve ADA although it is an adenosine-degrading enzyme. Interestingly, the modes of adenine salvage (Table 2) are not affected at all by ADA, AK or purine nucleoside phosphorylase (PNPase) deficiencies. That is, these modes do not require these enzymes. How- ever, they do require ADPRT, which is in agreement with the experimental observation mentioned in the Introduction that patients deficient in ADPRT are accumulating adenine [8–11]. The modes of adenosine salvage (Table 3) all require AK, so that they are not operative in the case of AK deficiency. This is clear because phosphorylation of adenosine is important in the buildup of ATP from adenosine. Five out of 12 modes require ADA, AK and PNPase, and another three require AK and PNPase but not ADA. None of the 12 modes requires ADPRT. The modes of ATP buildup in the presence of SAHH1 (but not SAHH2) and methyltransferase (Table 4) all require AK but not ADPR transferase. Six out of 12 modes require ADA, AK and PNPase and another three require AK and PNPase but not ADA. The modes in the presence of SAHH2 and MT (Table 4) do not require AK, while they do require ADPRT, in agreement with experimental findings [9,10]. Interestingly, the pathways using SAHH2 but not SAHH1 are completely independent of the three enzymes ADA, AK and PNPase. Out of the 10 modes involving SAHH but not methyl- transferase (Table 5), three modes do not require any of the enzymes ADA, AK and PNPase, the remaining seven require ADA and PNPase. AK is not required in any of the 10 modes. Interestingly, in these modes, it makes no difference whether ADA or PNPase are dele- ted, that is, a single deficiency in either enzyme has the same effect as the double deficiency. By contrast, in the modes of adenine salvage and adenosine salvage, dele- tion of PNPase is, on average, more critical than dele- tion of ADA. From Tables 2–5, it can easily be seen which elementary modes remain in the case of double or multiple deficiencies. For example, elementary mode 1 in Table 2 is still operating if ADA, AK and PNPase are deficient. In agreement with biochemical knowledge on human erythrocytes, HGPRT is not involved in any of the computed elementary modes corresponding to salvage pathways. Thus, hypoxanthine is not relevant for ATP salvage in these cells. Conclusions We have analysed, by mathematical modelling, the ATP buildup via salvage pathways in erythrocytes. Several authors used kinetic modelling to analyse erythrocyte metabolism [1,2,4]. We have used meta- bolic pathway analysis, which is a structural approach not requiring the knowledge of kinetic parameters. Pathway analysis has been applied to various enzyme deficiencies in the energy metabolism of erythrocytes [6] and to glutathione metabolism in a number of cells including erythrocytes [23]. Our results show once again that pathway analysis allows one to derive inter- esting conclusions about biochemical systems from a fairly limited amount of input information. The disad- vantage is that dynamic effects cannot be analysed. When different disease states are to be studied, the metabolite levels at different time scales need to be considered. In that case, a dynamic model is preferable [2]. Earlier, we had calculated the elementary modes in a subnetwork involving the enzymes of nucleotide metabolism only [24]. One of the elementary modes obtained corresponds to part of an adenine salvage pathway. The system studied here is much more exten- ded in that it involves glycolysis and the pentose phos- phate pathway in addition. We have found four elementary modes producing ATP starting from adenine. They involve parts of glycolysis and the pentose phosphate pathway in dif- ferent proportions. As far as the pentose phosphate pathway is concerned, there is some interrelation to the modes found earlier for that system [14]. In partic- ular, mode 1 (Table 2), which involves the oxidative pentose phosphate pathway and the enzyme R5PI, corresponds to the mode shown in Fig. 2D in Schuster et al. [14]. The modes II 2–4 correspond to the modes depicted in Fig. 2B,C,E, respectively [14]. However, R5PI is more active to provide the ribose necessary for ATP buildup. Twelve pathways of ATP buildup from adenosine have been found. However, only three of these convert adenosine completely into ATP. The other nine trans- form some of it to hypoxanthine to obtain free energy. Thus, the latter cannot be considered as perfect salvage pathways. They also serve the purpose of purine trans- port by erythrocytes [25]. Our results predict that there is redundancy both in adenine salvage and in adenosine salvage in that paral- lel pathways producing ATP from each of these sub- strates exist. While the metabolism of many cells is known to be redundant, this is surprising because erythrocyte metabolism in general has little redundancy and robustness. Earlier, we compared the structural S. Schuster and D. Kenanov A theoretical study using elementary flux modes FEBS Journal 272 (2005) 5278–5290 ª 2005 FEBS 5287 [...]... found that this route is formed by a set of 11 slightly different pathways (Table 4) We found, second and additionally, third parts 12 pathways starting from S-adenosylmethionine involving the standard functionality of SAHH (here denoted as SAHH1) and another 10 pathways starting from adenosine (rather than S-adenosylmethionine) and involving SAHH1 in the backward direction and SAHH2 in the forward direction... a novel result because these pathways do depend on AK (whereas Simmonds and coworkers [8– 11] only spoke about a pathway independent of AK) Interestingly, from Tables 4 and 5, it can be seen that the modes involving SAHH1 and ⁄ or SAHH2 do not depend on ADPR transferase if they involve AK and vice versa On the basis of elementary flux modes analysis, it can be said that, even though not easily provable... cover the complete erythrocyte metabolism The choice of reactions was motivated mainly by earlier models, textbook knowledge about salvage pathways and energy metabolism, as well as our aim to analyse the pathways using S-adenosylmethionine Regarding sensitivity of the model results to addition of enzymes, it is important that elementary modes have the favourable property that the set of elementary modes. .. genetic means, so that this drawback does not apply to such cells In metabolic pathway analysis, usually a distinction is made between internal and external metabolites Internal metabolites are intermediates that have to fulfil a balance equation at steady state, that is, their production must equal their consumption External metabolites are the sources and sinks of the network and are assumed to have buffered... step leading from IMP to AMP [8] From our theoretical analysis, a hitherto rarely discussed feature of the salvage pathways becomes transparent and understandable This is the high number of ATP molecules degraded in some part of each pathway while the total balance of ATP production is positive A ‘molar investment ratio’ could be defined to express the number of moles of ATP consumed divided by the difference... build up ATP by salvage pathways It has sometimes been suggested that, if parallel pathways exist, living cells use the pathway with the highest yield [27] or obeying a minimum flux criterion [5] It will be interesting to analyse, in the future, which 5288 S Schuster and D Kenanov of the salvage pathways are preferably used in vivo and whether they comply with these criteria This, however, is beyond the. . .A theoretical study using elementary flux modes robustness of Escherichia coli and erythrocytes and found that the latter is less robust [19] In glycolysis, deletion of one enzyme (e.g hexokinase) may suppress the entire pathway Therefore, hexokinase or phosphofructokinase deficiencies have severe consequences [26] Here, we have shown that the salvage pathways have a relatively high redundancy This... the scope of the present study, which is aimed at enumerating all potential pathways Simmonds and coworkers [8–11] proposed a novel route of ATP synthesis starting from S-adenosylmethionine or other nucleoside analogues That route involves SAHH and is independent of AK but dependent on ADPRT We have examined whether this way of ATP buildup is stoichiometrically and thermodynamically feasible The result... mode 1 of adenine salvage (Table 2), this ratio is 18:(20–18) ¼ 9 : 1 Consider, for comparison, the glycolytic pathway Two ATP are invested at the upper end of the pathway while four ATP are gained in the process, so that the difference is two The molar investment ratio is one (2 : 2) In all salvage pathways found here, this ratio is much higher Thus, a considerable effort in terms of enzyme activity... S-adenosylhomocysteine hydrolase 5289 A theoretical study using elementary flux modes 13 14 15 16 17 18 19 20 21 22 23 by nucleoside analogs Arch Biochem Biophys 207, 175– 184 Palmer JL & Abeles RH (1979) The mechanism of the action of S-adenosylhomocysteinase J Biol Chem 254, 1217–1226 Schuster S, Fell DA & Dandekar T (2000) A general definition of metabolic pathways useful for systematic organization and analysis of . Adenine and adenosine salvage pathways in erythrocytes and the role of S-adenosylhomocysteine hydrolase A theoretical study using elementary flux modes Stefan Schuster and Dimitar Kenanov Department. pathways using adenosine, a straightforward assumption is that these pathways start with adenosine kinase. However, a pathway bypassing this enzyme and using S-adenosylhomocysteine hydrolase instead was. pathways. Since adenine is a substrate of ADPRT, the elevation of ATP in the absence of adenosine kinase shows that adenine must be released in the process before being incorporated into ATP. Indeed,