Studies on Erythrocyte Glycolysis. Free Energy Changes and Rate Limitings Steps in Erythrocyte Glycolysis*,**

Transcription

1 The Journal of Biochemistry, Vol. 59, No. 2, 1966 II. Studies on Erythrocyte Glycolysis Free Energy Changes and Rate Limitings Steps in Erythrocyte Glycolysis*,** By SHIGEKI MINAKAMI and HARUHISA YOSHIKAWA (From the Department of Physiological Chemistry and Nutrition, Faculty of Medicine, the University of Tokyo, Bunkyo-ku, Tokyo) (Received for publication, September 10, 1965) The discussions on rate limiting steps in an enzymatic system inside cells have sometimes been done based on the estimations of enzymatic activities of the individual enzymes responsible for reactions. Usually, however, even the lowest activity of the enzyme in a system exceeds the overall activity of the system many-fold and it is not definite whether the step catalyzed by the enzyme of the lowest activity is actually the rate limiting step in cells. In erythrocytes, the lowest activity of glycolytic enzymes in hemolysate is that of hexokinase [EC ] and several investigators suggested that hexokinase is the rate limiting enzyme in the glycolysis (3, 4). If hexokinase is the only rate limiting step in glycolysis, no detectable glycolytic intermediates should be found inside the cells. All the glycolytic intermediates except 1, 3-diphospho- * Similar calculations based on different conditions of red cells have been published as a preliminary note (/).. The previous publication of this series was on the determination of the glycolytic intermediates and the nucleotides of red cells (2). ** The following abbreviations are used : G6P glucose 6-phosphate; F6P fructose 6-phosphate; FDP fructose 1,6-diphosphate; DHAP dihydroxyacetone phosphate GAP glyceraldehyde 3-phosphate; 1.3DPG 1,3-diphosphoglycerate; 2,3DPG 2, 3-diphosphoglycerate; 3PG 3-phosphoglycerate; 2PG 2-phosphoglycerate; PEP phosphoenolpyruvate. 139 glycerate were found in erythrocytes under physiological conditions. The logical way to obtain information on the intracellular activities of enzymes should be the investigation of whole cells. Hess (5) discussed on the control of glycolysis in intact ascites tumor cells by measuring the glycolytic intermediates in the cells and comparing cellular steady-state mass action ratios with the equilibrium constants of reactions. Human erythrocytes- seem to be one of the most suitable cells for the study of glycolytic reactions among living cells, as no nuclei, mitochondria nor endoplasmic reticulum are found inside the cells and glycolytic reaction can be regarded as taking place only in a homogeneous cytoplasma, so that ambigous assumptions such as " compartmentations to particulate fractions" may be unnecessary. The cells have no tricarboxylic acid cycle and respiration. Furthermore, hexose-monophosphate shunt contributes little to the break down of glucose in physiological conditions of the cells and both protein and lipid syntheses could not be observed in this sense. In the present study, free energy change in every step of glycolysis of erythrocytes was calculated on the basis of the intracellular concentrations of the glycolytic intermediates and the nucleotides of the cells which were reported in the previous communication (2). The advantages of the present calculations are 1) that the data are based on the concentrations of the reactants in the cells and 2) that there exists little interaction between glycolysis and other metabolic systems, such

2 140 S. MINAKAMI and H. YOSHIKAWA as respiration, hexose-monophosphate shunt and biosynthetic reactions. Rate limiting steps in the glycolytic reaction of erythroeytes in physiological condition will be discussed from these calculations. Intracellular Condition of Erythroeytes Employed for Thermodynamic Calculations The contents of the glycolytic intermediates and the nucleotides in the cells are those determined for human red cells which were analyzed immediately after removal from a cubital vein (2). The means of five separate determinations were used. The intracellular concentrations were calculated on the assumption that the water content inside the cells is 0.7 ml./ml. cells. It was assumed that glucose, lactate, pyruvate and inorganic phosphate were evenly distributed between the cells and plasma. These assumptions may be justified as the substances are known to be passively transported through erythrocyte membrane. The intracellular ph of erythroeytes in physiological condition is assumed to be ph7.0 according to Donnan equilibria (3, 6). The ratio [NAD + ]/[NADH] was calculated from the TABLE I Conditions of Erythroeytes Employed for the Calculations Glucose G6P F6P FDP DHAP GAP 1,3 DPG 2,3 DPG 3PG 2PG PEP Pyruvate Lactate ATP ADP Pi [NAD+]/[NADH] Intracellular ph 1) Calculated. 5 mm iim fim 7 nm 17 fiai 5. 7 fim 0. 4 fim " 5. 7 mm fim 10 fim 17 um 85 ^M 1.43mM 1.83mAf 180 fim 1.0 mm 985'> 7.0 [Lactate]/[Pyruvate] ratio and the standard free energy change of lactate dehydrogenase [EC ] at ph7.0 (<9), based on the conclusion of Klingenberg and Bucher (7) that the steady - states of the NAD + - dependent reactions in cytoplasma are in states of quasi-equilibria. The ratio of free [NAD + ]/[NADH] thus calculated was 985. The data used for thermodynamic calculations are tabulated in Table I. Calculations of Mass Action Ratios Table II shows the mass action ratios of individual steps in erythrocyte glycolysis calculated from the data of Table I. The equilibrium constants of enzymatic reactions calculated from the standard free energy changes of glycolytic reactions according to Burton (<5), are also shown in the Table. It is apparent from these calculations, that the reactions catalyzed by glucosephosphate isomerase [EC ], aldolase [EC ], triosephosphate isomerase [EC ], glyceraldehydephosphate dehydrogenase [EC ], phosphoglycerate kinase [EC ], phosphoglyceromutase [EC ] and phosphopyruvate hydratase [EC ] are in states of quasiequilibria, while the reactions catalyzed by hexokinase, phosphofructokinase [EC ] and pyruvate kinase [EC ] are extremely displaced from equilibria. The reaction of lactate dehydrogenase was assumed to be in a quasi-equilibrium state (7). In the group of the reactions which are in states of quasi-equilibria, the reactions catalyzed by aldolase and triosephosphate isomerase are somewhat displaced from equilibria. This may be explained by the hypothesis of the complex formation of aldolase and triosephosphate isomerase and the compartmentation of dihydroxyacetone phosphate at molecular level proposed by Garfinkel (9). The mass action ratio calculated from the overall reaction catalyzed by aldolase and triosephosphate isomerase is found to be in a quasi-equilibrium state. The calculated data are in good agreement with those of Hess (5) for intact ascites tumor cells, except that the overall reaction catalyzed by glyceraldehydephosphate dehydrogenase and phosphoglycerate kinase

3 TABLE II Mass Action Ratios of the Glycolytic Reactions in Erythrocytes [G6P][ADP] [Glucose][ATP] [F6P] [G6PJ [FDP][ADP] [F6P][ATP] [GAPJ[DHAP] [FDP] [GAP] [DHAP] [GAP] 2 [FDP] [3PG][ATP][NADH] [GAP][ADP][Pi][NAD+] [2PG] [3PG] [PEP] -[2FG] [Pyruvate][ATP] [PEP][ADP] [Lactate][NAD + ] [Pyruvate][NADH] Glucose+ATP G6P >F6P F6P+ATP FDP '2GAP Studies on Erythrocyte Glycolysis. II 141 Mass action ratio 7.6X X10" 2 1.4X10" 5 3.5x10"' 4.65x10- I.24X Reaction 1.7 'G6P+ADP >FDP+ADP GAP+NAD + +ADP+Pi 3PG 2PG *2PG 'PEP PEP+ADP 1.6x10' (assumed) 'Pyruvate+ATP Pyruvate+NADH >Lactate+NAD + Equilibrium constant 4xlO X x10-* 5xlO x10-2x10= x 10' 1.6x10' TABLE III Free Energy Changes in Erythrocyte Glycolysis {Kcal) '3PG+NADH+ATP are in a quasi-equilibrium state in the present study. Free Energy Changes in Individual Reactions of Glycolysis Thermodynamic considerations on enzymatic reactions are often made on the basis of standard free energy changes of the reactions. Standard free energy change (AF') is defined as the value calculated for the condition that all the reaetants except hydrogen ion are at one molar concentrations. The intracellular concentrations of metabolic intermediates are usually far from such standard conditions. Burton and Krebs (W) pointed out the problem, though the lack of data at that time forced them to calculate the free energy changes on the assumption that the concentrations of all reaetants be lomm. As the concentrations of most of the reaetants are less than 0.1 mm (see Table I), it seemes to be necessary to calculate the free energy changes (AF) on the basis of the actual concentrations of the intermediates in the cells. Table III shows the free energy changes of the individual reactions in the glycolysis of erythrocytes in physiological conditions as calculated from the mass action ratios given -10 AF (assumed) AF/mole glucose AF' Glucose+2ADP+2Pi ATP 'ADP+Pi Glucose '2 Lactate >2Lactate+2ATP (-52. 5) DPG 2,3DPG '2.3DPG '3PG+Pi

4 142 S. IVfiNAKAMi ahd H. VOSHIKAWA in Table II, applying the equation &F= 1.42 log K (K representing the mass action ratio of a reaction). The standard free energy data are those of Burton (8) for the condition of ph 7.0 and 25 C. The changes in free energy due to temperature (25 to 37 C) are neglected as they are not so large {11). Free energy changes per mole of glucose (the second column of Table III) are also graphically shown in Fig. 1 to visualize the free energy change during glycolysis. It is apparent from the figure that most of free energy changes during erythrocyte glycolysis take place at the levels of hexokinasc, phosphofructokinasc and pyruvate kinasc. FDP T 10 kcal./mole glucose GAP Lactate X'IG. 1. The free energy changes during erythrocyte glycolysis. The free energy changes {&F) are expressed as kcal/mole glucose. Discussions on Rate Limiting Reactions in Erythrocyte Glycolysis If an enzymatic system is in a steady state, the localizations of rate limiting steps in the enzymatic sequence may be suggested by mass action ratios or free energy changes calculated from the concentrations of intermediates present. In the case of erythrocyte glycolysis, the concentrations of glucose and lactate are high and the glycolytic rate are linear for several hours, so that the glycolytic system in the cells can be regarded as being in a steady state. When a step in a steady state enzymatic system is found to be in a quasi-equilibrium state or in another word, when the free energy change of the step is small, it can be said with certainty that the activity of the enzyme catalyzing the reaction is strong enough compared with the overall activity of the enzymatic systems. Glucosephosphate isomerase, aldolase, triosephosphate isomerase, glyceraldehydephosphate dehydrogenase, phosphoglycer^te kinase, phosphoglyceromutase, phosphopyruvate hydratase and lactate dehydrogenase in erythrocytes under physiological conditions may belong to a group of enzymes, the activities of which are high enough compared with the overall rate of glycolysis. Even the lowest activity of these enzymes in hemolysate was found to be more than 50 times than the overall rate of the glycolysis. Three steps in the glycolysis which are catalyzed by hexokinase, phosphofructokinase and pyruvate kinase, respectively, are different from other steps mentioned above, in that their free energy changes (AF) are large, i.e., the reactions are extremely displaced from the equilibria. This means that the activities of these three enzymes are not strong enough compared with the overall rate of the glycolysis, so that the equilibrium of the reactants could not be attained. It may be said that these three steps catalyzed by hexokinase, phosphofructokinase and pyruvate kinase are rate limiting steps in erythrocyte glycolysis. It has sometimes been discussed that hexokinase is the rate limiting step in erythrocyte glycolysis (3, 4). However, in a system like glycolysis, it is difficult to assume that it involves only one rate limiting step. We should assume at least three steps hexokinase, phosphofructokinase and pyruvate kinase as rate limiting steps, from the present calculations. These three steps are known to be controlled by the levels of several intermediates and nucleotides, e.g. hexokinase by glucose 6-phosphate and inorganic phosphate, phosphofructokinase by fructose 6-phosphate, ATP, ADP, AMP, inorganic phosphate and pyruvate kinase by ATP. 1,3-Diphosphoglycerate and 2,3-Diphosphoglycerate The content of 1,3-diphosphoglycerate in red cells could not be determined because of the lability of the ester in acid and the probable low concentration inside

5 Studies on Erythrocyte Glycolysis. II 143 the cells. The concentration of 1,3-diphosphoglycerate in the cells may be calculated by assuming that it is equilibrated with glyceraldehyde 3-phosphate or 3-phosphoglycerate. This could be justified as the intraeellular concentration of 1,3-diphosphoglycerate was estimated to be 0.49 fim from the equilibrium constant of glyceraldehydephosphate dehydrogenase and to be 0.31 pm from that of phosphoglycerate kinase. The concentration is too small for the assay method employed. The concentration of 2,3-diphosphoglycerate in erythrocyte is extremely high compared with that in other cells and the possibility of the pathway: 1,3-diphosphoglycerate " 2,3- diphosphoglycerate * 3 - phosphoglycerate+pi, has been suggested by several investigators (12). If we assume that the conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate takes place exclusively via this route, the free energy changes due to the reaction of 1,3-diphosphoglycerate to 2,3-diphosphoglycerate and that of 2,3-diphosphoglycerate to 3-phosphoglycerate are calculated to be 3.45 kcal and 11 kcal, respectively*. These reactions should also be rate limiting steps in the glycolysis, if the glycolysis exclusively takes the way through this shunt. This shunt, however, may be operating in steady state glycolysis of the cells in physiological condition only a limited extent and may be disregarded, since the rate of P 32 incorporation into 2,3-diphosphoglycerate is several times smaller than that into ATP (13). Efficiency of ATP Formation in Erythrocytes The intraeellular free energy change associated with the hydrolysis of ATP depends on the concentrations of ATP, ADP and inorganic phosphate in cells. The change in red cells can be calculated from the values * The free energy changes were calculated from the standard free energy data for the hydrolysis of 1,3-diphosphoglycerate and 2-phosphoglycerate( kcal (5) and 4 kcal (15), respectively) with the assumption that the intraeellular concentration of 1,3-diphosphoglycerate be 0.4 fim. The free energy change for 2-phosphoglycerate»glycerate+Pi was assumed to be identical with that for 2,3-diphosphoglycerate»3-phosphogIycerate+Pj. given in Table I as 14.3 kcal assuming the standard free energy change of ATP 2 " " ADP- + HPO l-h + to be -8.6 kcal at ph 7 (in The free energy change for the erythrocyte glycolysis may be calculated to be 52.5 kcal, using the value 47.4 kcal as the standard free energy change of glycolysis. It can also be calculated to be 53.8 kcal from the sum of the free energy changes for one mole of glucose converting into lactate ( 25.2 kcal: the sum of the values in the second column of Table III) and of the free energy change for the hydrolysis of 2 moles of ATP (-28.6 kcal). The efficiency of ATP formation by the steady state glycolysis of erythrocytes is thus calculated to be about 50%. SUMMARY 1. The mass action ratios and the free energy changes of the individual steps of glycolysis occurring in erythrocytes under physiological conditions are calculated. 2. The thermodynamic data indicate that three reactions catalyzed by hexokinase, phosphofructokinase and pyruvate kinase are rate limiting steps in glycolytic process in erythrocyte. 3. On the basis of the free energy change associated with the intraeellular hydrolysis of ATP, the efficiency of ATP formation in eythrocyte glycolysis was calculated. REFERENCES (/) Minakami, S., and Yoshikawa, H., Biochem. Biophys. Research Communs., 18, 345 (1965) (2) Minakami, S., Suzuki, C, Saito, T., and Yoshikawa, H., J. Biochem., 58, 543 (1965) (3) Hinterberger, U., Ockel, E., Gerischer-Mothes, W., and Rapoport, S., Ada Biol. Med. German., 7, 50 (1961) (4) Chapman, R.G., Hennessey, M.A., Watersdorph, A.M., Huennekens, F.M., and Gabrio, B.W., J. Clin. Invest.,41, 1249 (1962) (5) Hess, B., " Funktionelle und Morphologische Organization der Zelle " Springer Verlag, Berlin, p. 163 (1962) (6") VanSlyke, D.D., Hastings, A.B., Murray, CD., and Sendroy, J., J. Biol. Chem., 65, 701 (1925) (7) Klingenberg, M., and Biicher, T., Ann. Reu. Biochem., 29, 669 (1960)

6 144 S. MINAKAMI and H. YOSHIKAWA (8) Burton, K., Erg. Physiol., 49, 275 (1957) (9) Garfinkel, D., Ann. N.Y. Acad. Sci., 108, 293 (1963) (10) Burton, K., and Krebs, H., Biochem. J., 54, 94 (1953) (//) Huennekens, F.M., and Whiteley, H.R., " Comparative Biochemistry ", ed. by M. Florkin and H.S. Mason, Academic Press, N.Y., Vol. I, p. 107 (1960) (12) Rapoport, S., Dietze, F., and Sauer, G., Ada Biol. Med. German., 13, 693 (1964) (13) Tatibana, M., Miyamoto, K., Odaka, T., and Nakao, M., J. Biochem., 48, 685 (1960) (14) Burton, K., " Biochemists' Handbook ", ed. by C. Long, E. and F. Spon, London, p. 94 (1961) (15) Atkinson, M.R., and Morton, R.K., " Comparative Biochemistry ", ed. by M. Florkin and H.S. Mason, Academic Press, N.Y., Vol. II, p. 1 (1960)